Multiple antigen presenting immunogenic composition, and methods and uses thereof

ABSTRACT

The present embodiments provide for an immunogenic multiple antigen presenting system comprising a polymer to which antigens are associated by complementary affinity molecules. For example, the polymer can be a polysaccharide, or antigenic polysaccharide, to which protein or peptide antigens from the same or different pathogens are indirectly linked. The present immunogenic compositions can elicit both humoral and cellular immune responses to one or multiple antigens at the same time.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. § 371 National Phase EntryApplication of International Application No. PCT/US2012/037412 filed May11, 2012, which designates the U.S., and which claims benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 61/484,934 filed May11, 2011, of U.S. Provisional Application No. 61/608,168 filed Mar. 8,2012, and of U.S. Provisional Application No. 61/609,974 filed Mar. 13,2012, the contents of each of which are incorporated fully herein byreference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAI067737 and AI051526 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 18, 2013, isnamed 701039-069664-US_SL.txt and is 9,177 bytes in size.

FIELD OF THE INVENTION

The present invention relates to molecular genetics, immunology, andmicrobiology. The present application is generally directed tocompositions and methods for preparation of immunogenic compositions.More specifically, an embodiment of the present invention provides foran immunogenic macro-complex comprising at least one protein or peptideantigen attached to a polymer, such as a polysaccharide, which may alsobe an antigen. In some embodiments, this complex can be used as animmunogenic composition, such as a vaccine, to confer a synergistichumoral and cellular immune response; and in some embodiments, elicitssynergistic antibody- and cell-mediated protection against pathogens,e.g., lethal infection and the mucosal carriage of such pathogens.

BACKGROUND OF INVENTION

Vaccines provide prevention of and treatment for a variety of diseases,including microorganism infection, viral infection, and cancers. Currentpolysaccharide based vaccines, however, are not always effective in themost vulnerable populations. For example, Streptococcus pneumonia(pneumococcus) and Salmonella typhi infections are two major diseasesfor children in developing countries. For typhoid fever, licensed Vipolysaccharide vaccines are ineffective in children under two-years-old.Nevertheless, the success of polysaccharide-based vaccines and passiveimmunization for the prevention of colonization or disease hasdemonstrated the importance of capsular antibodies, in particular incontrolling disease caused by S. pneumoniae. Further, studies in bothanimals and humans demonstrate that antibodies elicited frompneumococcal vaccination can protect against nasopharyngeal (NP)pneumococcal colonization, which precedes pneumococcal disease.

A limitation of the current polysaccharide pneumococcal vaccines is thatprotection by anticapsular antibody is limited by its serotypespecificity. For example, although the 7-valent pneumococcal conjugatevaccine (PCV7) has significantly reduced the incidence of invasivepneumococcal disease due to vaccine-type (VT) strains, recent studieshave shown that non-VT serotypes are gradually replacing VT pneumococcalpopulations, potentially limiting the usefulness of the vaccine. Thishas led to the evaluation of whether pneumococcal colonization can beprevented by immunization with conserved antigens. In particular,several pneumococcal proteins have been evaluated as vaccine candidatesin animal models of pneumococcal colonization. Mucosal immunization withsome of these proteins has been shown to elicit systemic and mucosalantibodies and to confer protection against pneumococcal disease andcolonization. There remains a need for an immunogenic composition thatincludes both pneumococcal polysaccharides and proteins, capable ofraising both robust cellular and humoral immune responses to allpneumococcal serotypes.

Additionally, the innate immune response provides rapid and usuallyeffective defense against microbial pathogens. This response involvescellular recognition of pathogen-associated molecules, triggeringproduction and release of inflammatory mediators, recruitment ofleukocytes, and activation of antimicrobial effectors. The Toll-likereceptors (TLRs), of which at least eleven have been described formammals, are capable of discriminating among a wide variety ofpathogen-associated molecules and eliciting protective responses. Forexample, TLR4 recognizes many microbial products, including those fromgram-negative bacteria, the F protein of respiratory syncytial virus,and cholesterol-dependent cytolysins (CDC) of gram-positive bacteria.Additionally, TLR2 recognizes a large number of microbial and syntheticcompounds. Thus, inclusion of such TLR agonists may enhance the immuneresponse to vaccines. There remains a need to improve the efficacy ofvaccines by eliciting an innate immune response (TLR-mediated or other)against infections such as pneumococcal colonization and disease.

SUMMARY OF THE INVENTION

The present invention provides for an immunogenic multiple antigenpresenting system (MAPS), useful for the production of immunogeniccompositions, such as those useful in vaccines. In particular, thepresent invention relates to compositions comprising an immunogeniccomplex comprising at least one type of polymer, e.g., a polysaccharide,that can, optionally, be antigenic; at least one antigenic protein orpeptide; and at least one complementary affinity-molecule paircomprising (i) a first affinity molecule that associates with thepolymer, and (ii) a complementary affinity molecule that associates withthe protein or peptide; such that the first and complementary affinitymolecules serve as an indirect link between the polymer with theantigenic protein or peptide. Accordingly, the polymer can attach atleast 1, or at least 2, or a plurality of the same or different proteinor peptide antigens. In some embodiments, the polymer is antigenic,e.g., the polymer is a pneumococcal capsular polysaccharide. In someembodiments, the protein or peptide antigens are recombinant protein orpeptide antigens.

The immunogenic compositions as disclosed herein can elicit both humoraland cellular responses to one or multiple antigens at the same time. Theimmunogenic compositions provide for a long-lasting memory response,potentially protecting a subject from future infection. This allows fora single immunogenic composition that raises a high titer of functionalanti-polysaccharide antibody, and is similar or compares favorably withthe antibody level induced by conventional conjugate vaccine. Moreover,there is no restriction to specific carrier protein, and various antigenproteins can be used in MAPS construct to generate a robustanti-polysaccharide antibody response. Additionally, the strong antibodyresponse and Th17/Th1 responses are specific to multiple proteinantigens presented via the MAPS composition. This presents a majoradvantage, as a means for eliciting two forms of immunity with oneconstruct. In addition to a more conventional immune response to anantigenic polysaccharide conjugated to a protein carrier, the presentinvention provides for a T-cell response and, more specifically, Th17and Th1 responses to proteins injected systemically. Moreover, thepresent immunogenic composition can incorporate ligands onto the polymerbackbone. This provides a potential to enhance specific B-cell or T-cellresponses by modifying protein/polymer ratio, complex size, or byincorporating specific co-stimulatory factor, such as TLR2/4 ligands,etc., into the composition.

Compared with typical conjugation technology, which involves harshtreatment of proteins, the present methods avoid risk of denaturation ofother modification of the peptide antigen. This provides a substantialadvantage of preserving the antigenicity of the included proteins andincreases the probability that the protein itself will serve as anantigen (rather than just a carrier). Similarly, the present methodsavoid unnecessary modification/damage of the polysaccharide backbone,because there is no heavy chemical cross-linking: biotinylation can beprecisely controlled to react with specific functional groups of thepolysaccharide, and the biotinylation level can be easily adjusted. Thisis advantageous in avoiding the typical process of conjugation, thatresults in damage to critical side chains or epitopes, which may causereduced immunogenicity and protection.

The present the affinity-based assembly provides easy and highlyflexible preparation of the immunogenic composition. It is highlyspecific and stable; it can remain in the cold for months and retain itspotency. The assembly process is simple enough to ensure highreproducibility; there are only a few steps required, which reduces therisk of lot-to-lot variation, of great industrial advantage. The MAPSassembly is highly efficient (over 95%), even at low concentrations ofprotein and polysaccharide (such as 0.1 mg/ml); this is a majoradvantage, because inefficiencies in conjugate manufacture (typicallyefficiencies are in the <50% range) represent a major hurdle and reasonfor the high cost of vaccines. For formulation: it is easy to adjust thecomposition and physical properties of the final product. Theprotein:polymer ratio in the complex is adjustable; with moderatebiotinylation of polymer, protein:polymer can be 10:1 (w/w) or more;conversely, the ratio can be 1:10 or less if such is the interest basedon immunological goals. Additionally, the size of the immunogenic MAPScomposition can be adjusted by the choice of polymer size. The methodsof making the MAPS provide for ease in combining proteins and polymerswith little modification. The possible multivalency of final product byloading multiple protein antigens, from the same or different pathogens(e.g., pneumococcus and tuberculosis), in single immunogenic construct,provides for a composition that can be used to decrease the number ofvaccines required to immunize a subject against more than one disease.Moreover, the MAPS composition is highly stable; becoming dissociatedonly upon boiling and maintaining immunogenicity even after many monthsat 4° C. The immunogenicity of the MAPS complex may be limited by thestability of the antigenic protein or peptide component, which stabilitymay be extended by inclusion in the MAPS complex. The specific antigensused herein exhibited stability at room temperature and after at leastone freeze-thaw cycle. This provides an important advantage over currentvaccines that are compromised if the “cold chain” is not maintainedcarefully.

Accordingly, one aspect of the present invention relates to animmunogenic composition comprising a polymer, at least one protein orpeptide antigen, and at least one complementary affinity-molecule pair,where the complementary affinity-molecule pair comprises a firstaffinity molecule that associates with the polymer and a complementaryaffinity molecule that associates with the protein or peptide antigen,so that when the first affinity molecule associates with thecomplementary affinity molecule, it indirectly links the antigen to thepolymer.

In some embodiments, the first affinity molecule is cross-linked to thepolymer with a cross-linking reagent, for example, a cross-linkingreagent selected from CDAP(1-cyano-4-dimethylaminopyridiniumtetrafluoroborate), EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride), sodium cyanoborohydride; cyanogen bromide; or ammoniumbicarbonate/iodoacetic acid. In some embodiments, the first affinitymolecule is cross-linked to carboxyl, hydroxyl, amino, phenoxyl,hemiacetal, and mecapto functional groups of the polymer. In someembodiments, the first affinity molecule is covalently bonded to thepolymer.

In some embodiments, the first affinity molecule is biotin or aderivative thereof, or a molecule with similar structure or physicalproperty as biotin, for example, an amine-PEG3-biotin((+)-biotinylation-3-6,9-trixaundecanediamine) or derivative thereof.

In some embodiments, the protein or peptide antigen of the immunogeniccomposition is a fusion protein comprising the antigenic protein orpeptide fused to the complementary affinity binding molecule. The fusioncan be a genetic construct, i.e., a recombinant fusion peptide orprotein. In some embodiments, an antigen can be covalently attached as afusion protein to the complementary affinity molecule. In alternativeembodiments, the antigen is non-covalently attached to the complementaryaffinity molecule.

In some embodiments, the complementary affinity molecule is abiotin-binding protein or a derivative or a functional portion thereof.In some embodiments, a complementary affinity molecule is an avidin-likeprotein or a derivative or a functional portion thereof, for example butnot limited to, rhizavidin or a derivative thereof. In some embodiments,a complementary affinity molecule is avidin or streptavidin or aderivative or a functional portion thereof.

In some embodiments, a secretion signal peptide is located at theN-terminus of the avidin-like protein. Any signal sequence known topersons of ordinary skill in the art can be used; and in someembodiments, the signal sequence is MKKIWLALAGLVLAFSASA (SEQ ID NO:1) ora derivative or functional portion thereof. In some embodiments, theantigen can be fused to a complementary affinity molecule via a flexiblelinker peptide, where the flexible linker peptide attaches the antigento the complementary affinity molecule.

In some embodiments, the polymer component of the immunogen comprises apolymer derived from a living organism, e.g., a polysaccharide. In someembodiments, a polymer can be purified and isolated from a naturalsource, or is can be synthesized as with a naturalcomposition/structure, or it can be a synthetic (e.g., with anartificial composition/structure) polymer. In some embodiments, apolymer is derived from an organism selected from the group consistingof: bacteria, archaea, or eukaryotic cells like fungi, insect, plant, orchimeras thereof. In some embodiments, the polymer is a polysaccharidederived from a pathogenic bacterium. In specific embodiments thepolysaccharide is a pneumococcal capsular polysaccharide, a pneumococcalcell-wall polysaccharide, or a Salmonella typhi Vi polysaccharide.

In some embodiments, a polymer of the immunogenic composition asdisclosed herein is branched chain polymer, e.g., a branchedpolysaccharide, or alternatively, can be a straight chain polymer, e.g.,a single chain polymer, e.g., polysaccharide. In some embodiments, thepolymer is a polysaccharide, for example, dextran or a derivativethereof. In some embodiments, a polymer, e.g., dextran polysaccharidecan be of average molecular weight of 425kD-500 kDa, inclusive, or insome embodiments, greater than 500 kDa. In some embodiments, a polymer,e.g., dextran polysaccharide can be of average molecular weight of60kD-90 kDa, inclusive, or in some embodiments, smaller than 70 kDa. Thedextran polymer can be derived from a bacterium, such as Leuconostocmesenteroides.

In some embodiments, an immunogenic composition as disclosed hereincomprises at least 2 antigens, or at 3 least antigens, or at least 5antigens, or between 2-10 antigens, or between 10-15 antigens, orbetween 15-20 antigens, or between 20-50 antigens, or between 50-100antigens, or more than 100 antigens, inclusive. In some embodiments,where an immunogenic composition as disclosed herein comprises at least2 antigens, the antigens can be the same antigen or at least 2 differentantigens. In some embodiments, the antigens can be from the same ordifferent pathogens, or can be different epitopes or parts of the sameantigenic protein, or can be the same antigen which is specific todifferent serotypes or seasonal variations of the same pathogen (e.g.,influenza virus A, B, and C).

In some embodiments, an immunogenic composition as disclosed hereincomprises an antigen from a pathogenic organism or an abnormal tissue.In some embodiments, the antigen is a tumor antigen. In someembodiments, an antigen can be at least one antigen selected fromantigens of pathogens or parasites, such as antigens of Streptococcuspneumoniae, Mycobacterium tuberculosis or M. tetanus, Bacillusanthracis, HIV, seasonal or epidemic influenza antigens (such as H1N1 orH5N1), Bordetella pertussis, Staphylococcus aureus, Neisseriameningitides or N. gonorrhoeae, HPV, Chlamydia trachomatis, HSV or otherherpes viruses, or Plasmodia sp. These antigens may include peptides,proteins, glycoproteins, or polysaccharides. In some embodiments, theantigen is a toxoid or portion of a toxin.

In some embodiments, an immunogenic composition as disclosed hereincomprises an antigenic polysaccharide, for example, such as Vi antigen(Salmonella typhi capsular polysaccharide), pneumococcal capsularpolysaccharides, pneumococcal cell wall polysaccharide, Hib (Haemophilusinfluenzae type B) capsular polysaccharide, meningococcal capsularpolysaccharides, the polysaccharide of Bacillus anthracis (the causativeagent of anthrax), and other bacterial capsular or cell wallpolysaccharides, or any combinations thereof. The polysaccharide mayhave a protein component, e.g., a glycoprotein such as those fromviruses.

In some embodiments, an immunogenic composition as disclosed hereinfurther comprises at least one co-stimulation factor associated with thepolymer or polysaccharide, where the co-stimulation factor can beassociated directly or indirectly. For example, in some embodiment, aco-stimulation factor can be covalently attached to the polymer. Forexample, in some embodiments, a co-stimulation factor can be covalentlyattached to the first affinity molecule, which is then cross-linked tothe polymer. For example, in some embodiments, a co-stimulation factorcan be attached to a complementary affinity molecule, which associateswith a first affinity molecule to link the co-stimulation factor to thepolymer. In some embodiments, a co-stimulation factor is an adjuvant. Inalternative embodiments, a co-stimulatory factor can be any known to oneof ordinary skill in the art, and includes any combination, for example,without limitation, Toll like receptor agonists (agonists for TLR2, 3,4, 5, 7, 8, 9, etc.), NOD agonists, or agonists of the inflammasome.

Another aspect of the present invention relates to the use of theimmunogenic composition as disclosed herein to be administered to asubject to elicit an immune response in the subject. In someembodiments, the immune response is an antibody/B cell response, a CD4⁺T-cell response (including Th1, Th2 and Th17 cells) and/or a CD8⁺ T-cellresponse. In some embodiments, at least one adjuvant is administered inconjunction with the immunogenic composition.

Another aspect of the present invention relates to a method for inducingan immune response in a subject to at least one antigen, comprisingadministering to the subject the immunogenic composition as disclosedherein.

Another aspect of the present invention relates to a method ofvaccinating an animal, e.g., a bird, a mammal or a human, against atleast one antigen comprising administering a vaccine compositioncomprising the immunogenic composition as disclosed herein.

In all aspects as disclosed herein, an animal or a subject can be ahuman. In some embodiments, the subject can be an agricultural ornon-domestic animal, or a domestic animal. In some embodiments, avaccine composition comprising the immunogenic composition as disclosedherein can be administered via subcutaneous, intranasal, oral,sublingual, vaginal, rectal, intradermal, intraperitoneal, intramuscular injection, or via skin-patch for transcutaneous immunization.

In all aspects as disclosed herein, an immune response is anantibody/B-cell response, a CD4⁺ T-cell response (including Th1, Th2 andTh17 responses) or a CD8+ T-cell response against protein/peptideantigen(s). In some embodiments, an immune response is anantibody/B-cell response against the polymer, e.g., a pneumococcalpolysaccharide. In some embodiments, at least one adjuvant isadministered in conjunction with the immunogenic composition.

Another aspect of the present invention relates to the use of theimmunogenic composition as disclosed herein for use in a diagnostic forexposure to a pathogen or immunogenic agent.

Another aspect of the present invention relates to kits for preparing animmunogenic composition as disclosed herein. For example, such kits cancomprise any one or more of the following materials: a containercomprising a polymer, e.g., a polysaccharide, cross-linked with aplurality of first affinity molecules; and a container comprising acomplementary affinity molecule which associates with the first affinitymolecule, wherein the complementary affinity molecule associates with anantigen.

In another embodiment, the kit can comprise a container comprising apolymer, e.g., a polysaccharide; a container comprising a plurality offirst affinity molecules; and a container comprising a cross-linkingmolecule for cross-linking the first affinity molecules to the polymer.In some embodiments, the kit can comprise at least one co-stimulationfactor which can be added to the polymer. In some embodiments, the kitcomprises a cross-linking reagent, for example, but not limited to,CDAP(1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sodiumcyanoborohydride; cyanogen bromide; ammonium bicarbonate/iodoacetic acidfor linking the co-factor to the polymer or polysaccharide. In someembodiments, the kit further comprises a means to attach thecomplementary affinity molecule to the protein or peptide antigen, wherethe means can be by a cross-linking reagent or by some intermediaryfusion protein.

In some embodiments, the kit can comprise a container comprising anexpression vector for expressing a protein or peptide antigen-affinitymolecule fusion protein, for example, an expression vector forexpressing the protein or peptide antigen as a fusion protein with thecomplementary affinity molecule. In some embodiments, the vector canoptionally comprise a sequence for a linker peptide, wherein theexpression vector can expresses an antigen-complementary affinitymolecule fusion protein comprising a linker peptide located between theantigen and the affinity molecule.

In some embodiments, the kit can optionally comprise a containercomprising a complementary affinity molecule which associates with thefirst affinity molecule, wherein the complementary affinity moleculeassociates with a peptide/protein antigen. In some embodiments, the kitcan additionally further comprise a means to attach the complementaryaffinity molecule to the antigen, e.g., using a cross-linking regent asdisclosed herein or other intermediately protein, such as a divalentantibody or antibody fragment.

Provided herein also is a method of vaccinating a subject, e.g., amammal, e.g., a human with the immunogenic compositions as disclosedherein, the method comprising administering a vaccine composition asdisclosed herein to the subject.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of multiple antigen presenting system(MAPS). MAPS represents a novel platform of a complex immunogeniccomposition, that is made by attaching a number of protein antigens to apolysaccharide or polysaccharide antigen via a stable interaction of anaffinity pair, such as avidin-biotin pair. In one embodiment of the MAPScomplex, the protein antigens from one or different pathogens arerecombinantly fused to an avidin-like protein and expressed in E. coli.The polysaccharide backbone, which may be chosen from a variety ofpathogens, is biotinylated and/or cross-linked with or withoutco-stimulation factors using 1-Cyano-4-dimethylaminopyridiniumtetrafluoroborate (CDAP) or1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as anactivating reagent. A MAPS complex can be assembled readily by justmixing and incubating the purified fusion antigens, one or multiple, atthe desired ratio, with biotinylated polysaccharide. Assembled MAPScomplex can be purified/separated according to size by gel filtrationchromatography.

FIG. 2 shows exemplary examples of biotinylation of polysaccharide: thestructures of the biotin derivative, amine-PEG3-biotin (also known as(+)-biotinylation-3-6,9-trixaundecanediamine); the structure of CDAP;and the structure of EDC. Figure also shows a schematic for the methodof biotinylation of polysaccharides using CDAP as the activatingreagent, process (1) or using EDC as the activating reagent, process(2). Other procedures for biotinylation are encompassed in the methodsof the invention.

FIGS. 3A-3C show an embodiment of a recombinant rhizavidin andrhizavidin-antigen fusion protein. FIG. 3A shows a schematic of theconstruction of modified rhizavidin (upper (SEQ ID NOS 29-30,respectively, in order of appearance)) and rhizavidin-antigen fusionprotein (lower (SEQ ID NOS 29, 27 and 31, respectively, in order ofappearance)). All constructs were cloned into PET21b vector andtransformed into E. coli BL21 (DE3) strain for expression. FIG. 3B showsSDS-PAGE of purified recombinant rhizavidin (rRhavi). FIG. 3C showsSDS-PAGE of purified rhavi-antigen fusion proteins. Lane 1, rhavi-Pdt;lane 2, rhavi-PsaA; lane 3, rhavi-sp1733; lane 4, rhavi-sp1534; lane 5,rhavi-sp0435; lane 6, rhavi-sp1458; lane 7, rhavi-ESAT-6/Cfp10; lane8,rhavi-TB9.8/TB10.4; lane 9, rhavi-MPT64; lane 10, rhavi-MPT83.

FIGS. 4A-4C show the elution profile of an assembled example MAPS. FIG.4A MAPS was assembled by incubating 0.5 mg of purified rRhavi with 1 mgof biotinylated dextran 90 (BD90, average MW 60-90 KD) at 4° C.overnight and then applied to a superdex-200 column. Peak A and Peak Bindicated the eluted fractions containing MAPS complex, Peak C indicatedthe eluted fractions containing free rRhavi. FIG. 4B shows SDS-PAGE ofpeak fractions. All samples were boiled in SDS sample buffer with 10 mMDTT. FIG. 4C shows the stability of MAPS complex. Equal amounts ofsample were treated and then applied to SDS-PAGE. MAPS complex remainsintact even after treatment of SDS sample buffer containing reducingreagent (lane 1) and can only be broken after boiling, attesting to thestability of the association. Lane 1, MAPS treated with SDS samplebuffer containing 10 mM DTT, room temperature for 10 min; lane 2, MAPStreated with SDS sample buffer without DTT, boiled for 10 min; lane 3,MAPS treated with SDS sample buffer containing 10 mM DTT, boiled for 10min.

FIG. 5 shows assembly of MAPS complex at different temperature and atdifferent concentration of PS and protein antigen. MAPS complex can beeffectively assembled at a wide range of concentrations ofpolysaccharide (PS) or protein antigen (as low as 0.1 mg/ml). Theassembly can be done by overnight incubation at 4° C. (FIG. 5A) or at25° C. (FIG. 5B), depending on the stability of the antigens. Theassembly efficiency of MAPS complex can be estimated by running theassembling mixture through SDS-PAGE, with or without boiling the samplebeforehand. Without boiling treatment, the protein antigens that wereincorporated into MAPS complex stay on the PS and thus show up as bandsof very large molecular weight on the gel (MAPS/PS); only the unboundproteins would run lower on the gel and detected at the expectedmolecular weight of the antigen (monomer or dimer position). Bycomparison of the protein antigen band before and after boiling, thepercentage of the antigens assembled into MAPS complex could beestimated. In general, the assembling efficiency at 4° C. is greaterthan 85%, and at 25° C., it is close to 95%-99%.

FIG. 6 shows elution profiles of MAPS assembled with different ratios ofprotein vs. polysaccharide. 0.5 mg of purified rRhavi was incubatedovernight with either 1 mg, 0.5 mg or 0.1 mg of BD90, respectively, andthen applied to gel filtration chromatography using superdex 200 column.The MAPS complex assembled at higher ratio of protein vs. polysaccharideappeared to have higher molecular weight than the one assembled at lowerratio. Peak fractions containing MAPS complex for each sample (indicatedby arrows) were collected. The ratio of protein vs. polysaccharide inthe purified MAPS complex was measured and showed good correlation tothe input ratio.

FIG. 7 shows elution profiles of MAPS assembled with various sizes ofpolysaccharide. 0.5 mg of fusion antigen was incubated with 0.25 mgbiotinylated dextran with an average molecular weight of 425-500 KD(BD500), 150 KD (BD150) or 60-90 KD (BD90). The MAPS complex wasseparated using Superpose 6 column; the chromatography profile showedthat the complex assembled with a bigger polysaccharide had a largersize.

FIG. 8A-8D shows MAPS assembly with multiple antigens. FIG. 8A showsMAPS assembly with two antigens at different ratios. Bivalent MAPScomplex were prepared by incubating biotinylated S. pneumoniae (SP)serotype 14 capsular polysaccharide with two different pneumococcalfusion antigens rhavi-1652 and rhavi-0757 mixed at a molar ratio of 1:4,1:2, 1:1, 2:1, or 4:1. SDS-PAGE showed that the amounts of each antigenincorporated into the MAPS complex were well correlated to the inputratios. FIGS. 8B-8D show multivalent MAPS complex that were made withbiotinylated polysaccharide (dextran, or serotype 3 pneumococcalcapsular polysaccharide) connecting two (2V, FIG. 8B), three (3V, FIG.8C) or five (5V, FIG. 8D) different pneumococcal and/or tuberculosisantigens. SDS-PAGE showed the antigens incorporated into MAPS complex.All samples were boiled in SDS sample buffer with 10 mM DTT.

FIG. 9 shows that immunization with a MAPS complex induced a strongantibody response against polysaccharide antigens. Mice that wereimmunized with MAPS complex made from biotinylated dextran (9A), Vipolysaccharide (9B), or pneumococcal cell wall polysaccharide (CWPS)(9C) made a significant higher amount of anti-polysaccharide antibodiescompared to the animal groups that received adjuvant alone (no Ag) or amixture of uncoupled polysaccharide and proteins (Mixture). FIGS. 9D-9Fshow that MAPS complex compares favorably with conventional conjugatevaccine in generating anti-PS Ab. MAPS complexes were made from SPserotype 1, 5, 14 capsular polysaccharide (CPS), loaded with fiveprotein antigens. Mice were subcutaneously immunized with MAPS orPrevnar 13® (Pneumococcal 13-valent Conjugate Vaccine [Diphtheria CRM197Protein]; Wyeth/Pfizer) (PCV13) twice, 2 weeks apart, and the serum IgGantibody against vaccinated serotype CPS was analyzed 2 weeks after thesecond immunization by ELISA. The titer of anti-CPS IgG in PCV13immunized mice was arbitrarily set at 1200 units for comparison. For alltested serotypes, immunization with MAPS complex generated eithersimilar level (serotype 5) or much greater level of anti-CPS IgGantibody (serotype 1 and serotype 14) than what generated by vaccinationwith PCV13. Serotype 1 (FIG. 9D); Serotype 5 (FIG. 9E); Serotype 14(FIG. 9F).

FIG. 10 compares anti-PS antibody induced by MAPS at differentimmunization dosages. MAPS complex was made from serotype 5 SP CPSloading with five protein antigens. Mice were given with MAPS complex at1 μg-16 μg of PS content per dose, for two immunizations, two weeksapart. Serum antibody against serotype 5 CPS was measured and comparedbetween different immunization groups two weeks after the secondimmunization. At all dosages, immunization with MAPS induced robust IgGantibody against serotype 5 CPS. Giving 2 μg of PS per dose generatedthe highest antibody titer, and increasing PS dosage to 16 μg reducedthe antibody titer about 4-fold.

FIG. 11 shows that the anti-PS antibodies generated by immunization withMAPS complex facilitate the killing of the target pathogens in vitro.FIG. 11A demonstrates the antibody-mediated killing of the Vi-expressingbacterium. The serum from the animals immunized with MAPS complex (usingVi as the backbone), but not from the two other groups, showed potentkilling of the Vi-expressing strain (more than 90% killing) within 1hour of incubation. Serum from mice immunized with Alum (dashed line);Mixture (black line); or MAPS (gray line). FIGS. 11B-11D demonstratethat opsonophagocytic killing activity of serum from MAPS-immunized micecompares favorably to the killing activity of serum from mice immunizedwith licensed vaccine PCV13. The ability of the serum from PCV13- orMAPS-immunized mice in mediating in vitro opsonophagocytic killing ofpneumococcus by neutrophils was analyzed and compared. Human neutrophilswere differentiated from cells in the HL-60 cell line. Theopsonophagocytic killing was done by incubating the serum, in differentdilutions, with serotype 1 (FIG. 11B), serotype 5 (FIG. 11C) or serotype13 (FIG. 11D), pneumococcus and differentiated HL-60 cells at 37° C. for1 hour (in the presence of baby rabbit complement). An aliquot of themixture was plated after incubation for counting of the survivalbacteria. The opsonophagocytic killing unit was defined as the folddilution of the serum which 50% killing of the bacteria is observed. Forall tested serotypes, serum from MAPS immunized mice showed at least 4times higher killing activity (OPA titer) than serum from PCV13immunized mice. FIGS. 11B-11D: Serum from mice immunized with Alum(dashed line); PCV13 (black line); or MAPS (gray line).

FIGS. 12A-12D demonstrate that immunization with a MAPS complex inducesrobust antibody and cellular response against protein antigens. BivalentMAPS complex was made from biotinylated dextran (BD500) and twopneumococcal antigens, rhavi-Pdt and rhavi-PsaA. Subcutaneousvaccinations were given biweekly, three times. FIG. 12A shows theresults of serum IgG antibodies measured against PsaA or Pdt 2 weeksafter the last immunization. Mice immunized with MAPS complex madesignificantly higher titer of anti-Pdt and anti-PsaA antibodies thanmice that received the mixture. Antigen specific T-cell responses wereevaluated by in vitro stimulation of the whole blood of immunizedanimals. IL-17A (FIG. 12B) and IFN-γ (FIG. 12C) production in vitro wasmeasured in blood samples incubated 6 days with either purified PsaA,Pdt, or pneumococcal whole-cell antigen (WCA). Compared to the miceimmunized with the mixture, the animals that received MAPS complexshowed significantly stronger IL-17A and IFN-γ response. FIG. 12D showsa correlation of IL-17A and IFN-γ production by stimulation of WCA. Forall panels, bars represent means with standard deviation and statisticalanalysis was performed using Mann-Whitney test, or using Spearman R forcorrelation.

FIG. 13 shows the evaluation of the immunogenicity of MAPS complex indifferent sizes. MAPS complexes were made from two pneumococcal fusionantigens, rhavi-PsaA and rhavi-Pdt, and using dextran in differentlength as backbone (BD500, Mw of 425-500 kDa; BD90, Mw of 60-90 kDa).The antibody responses to dextran, and to two protein antigens PdT andPsaA, as well as the antigen specific T cell responses were measured andcompared after 3 immunizations. As shown, mice that were immunized withthe bigger complex (MAPS BD500) generated the similar level of anti-PsaAand anti-Pdt antibodies (FIG. 13B), but the significantly higher titerof anti-dextran antibody (FIG. 13A) as well as the IL-17A associated Tcell response (FIG. 13C) than animals received the smaller complex (MAPSBD90).

FIG. 14 shows that addition of co-stimulatory factors (TLR ligands) tothe MAPS complex facilitates the IL-17A and IFN-γ associated T cellresponses. MAPS complexes were made from biotinylated dextran and onepneumococcal protein antigen, rhavi-0435, with or without the additionalTLR ligand/agonist: rhavi-Pdt, TLR4 ligand; Pam3CSK4, TLR2 agonist. Theincorporation of rhavi-Pdt is via affinity interaction between rhavi andbiotin, whereas Pam3CSK4 is covalently linked to the dextran backbone.Immunization was given subcutaneously for three times, and the T cellresponses against 0435 protein were measured and compared. It showedthat addition of TLR2 agonist or a combination of TLR4 and TLR2 ligandssignificantly enhanced the IL-17A and IFN-γ associated T cell responsesto the protein antigen.

FIG. 15 shows an example of multivalent pneumococci/mycobacteriumtuberculosis (TB) combination vaccine. Multivalent SP/TB combinationMAPS vaccine was prepared by using SP serotype 3 and loading one SPprotein (pneumolysin toxoid, Pdt) and six TB proteins (in four fusionconstructs) (FIG. 15A). Immunization of mice with SP/TB MAPS induced agreat titer of IgG antibody to type 3 CPS (FIG. 15B, left panel), aswell as to Pdt (FIG. 15B, right panel), and led to 100% protection ofmice from fatal lung infection of serotype 3 pneumococcus (FIG. 15C).FIGS. 15D-15J show the B-cell and T-cell immunity B antigens induced byvaccination with SP/TB MAPS. FIG. 15D shows the antibody responses todifferent TB protein antigens. FIGS. 15E-15F show strong IL-17A (FIG.15E) and IFN-γ (FIG. 15F) associated T cell responses in whole bloodsample from MAPS immunized mice after in vitro stimulation with purifiedTB protein antigens. FIGS. 15G and 15H show the IL-17A (FIG. 15G) andIFN-γ (FIG. 15H) associated T-cell responses of splenocytes from MAPSimmunized animals to the mixture of purified TB protein antigens or tothe TB whole cell extract. FIGS. 15I and 15J provide further dataregarding the TB-specific memory/effector T-cells induced byimmunization with MAPS. The results showed that depletion of CD4+T-cells but not CD8+ T cells had a significant impact on the TB antigenspecific cytokine production, indicating that immunization with MAPSvaccine had primed mainly a CD4+ T-cell (T helper cell) immune response.

FIG. 16 demonstrates that a prototype MAPS-based multivalent immunogeniccomposition prevents invasive infection and nasopharyngeal colonizationof pneumococcus. Multivalent SP MAPS was made using SP cell wallpolysaccharide (CWPS) as the backbone and loaded with five pneumococcalprotein antigens (FIG. 16A). Mice were immunized with this SP MAPS threetimes, two weeks apart, and the serum antibodies and specific T-cellresponses against pneumococcus were analyzed two weeks after the lastimmunization. FIG. 16B shows serum IgG antibody against CWPS (leftpanel) or against pneumococcal whole cell antigen (WCA) (right panel).Mice immunized with SP MAPS made significant higher titer of antibodiesto either CWPS or WCA than mice in the control groups that receivedadjuvant alone (No Ag) or uncoupled PS/protein mixture (Mixture). FIGS.16C and 16D show SP-specific T-cell responses induced by vaccinationwith SP MAPS. Peripheral blood from mice of different immunizationgroups were stimulated with either purified pneumococcal proteins(antigen mix) or WCA. Cells from MAPS vaccinated mice but not from thecontrol groups responded to the SP antigens greatly and gave robustproduction of IL-17A (FIG. 16C) and IFN-γ (FIG. 16D). FIGS. 16E and 16Fshow that vaccination with MAPS complex protects mice from invasiveinfection as well as nasopharyngeal colonization of pneumococcus. Miceof different immunization groups were challenged either with SP serotype3 strain WU2 in a lung aspiration model (FIG. 16E), or with serotype 6pneumococcal strain 603 in a nasal colonization model (FIG. 16F).Protection against sepsis or colonization was only observed inMAPS-immunized mice.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.The term “or” is inclusive unless modified, for example, by “either.”Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

The present invention relates immunogenic compositions and compositionscomprising an immunogenic complex that comprises at least one antigen,or multiple antigens, attached to a polymer scaffold for use ineliciting an immune response to each of the antigens attached to thepolymer, and optionally to the polymer itself, when administered to asubject. This multiple antigen presenting system (MAPS), stimulates ahumoral and cellular immune response: it can generateanti-polysaccharide antibody and the B-cell/Th1/Th17 responses tomultiple protein antigens using single MAPS immunogenic construct. Acombination of B- and T-cell immunity to the organism might represent anoptimal vaccine strategy against many diseases, including pneumococcaldisease associated invasive infection and nasopharyngeal carriage. Insome embodiments, the immunogenic composition is a vaccine or isincluded in a vaccine.

Accordingly, one aspect of the present invention relates to animmunogenic composition (multiple antigen presenting system, or MAPS)comprising at least one polymer, e.g., one polysaccharide, at least oneprotein or peptide antigen, and at least one complementaryaffinity-molecule pair comprising (i) a first affinity moleculeassociated with the polymer, and (ii) a complementary affinity moleculeassociated with the antigen, which serves to indirectly attach theantigen to the polymer (e.g., the first affinity molecule associateswith the complementary affinity molecule to link the antigen to thepolymer). Accordingly, as the polymer can be used as a scaffold toattach at least 1, or at least 2, or a more (e.g., a plurality) of thesame or different antigens. The immunogenic compositions as disclosedherein can be used to elicit both humoral and cellular immunity tomultiple antigens at the same time.

Accordingly, the embodiments herein provide for an immunogeniccomposition and methods useful for raising an immune response in asubject, which can be used on its own or in conjunction or admixturewith essentially any existing vaccine approaches.

DEFINITIONS

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

The term “immunogenic composition” used herein is defined as acomposition capable of eliciting an immune response, such as an antibodyor cellular immune response, when administered to a subject. Theimmunogenic compositions of the present invention may or may not beimmunoprotective or therapeutic. When the immunogenic compositions ofthe present invention prevent, ameliorate, palliate or eliminate diseasefrom the subject, then the immunogenic composition may optionally bereferred to as a vaccine. As used herein, however, the term immunogeniccomposition is not intended to be limited to vaccines.

As used herein, the term “antigen” refers to any substance that promptsan immune response directed against the substance. In some embodiments,an antigen is a peptide or a polypeptide, and in other embodiments, itcan be any chemical or moiety, e.g., a carbohydrate, that elicits animmune response directed against the substance.

The term “associates” as used herein refers to the linkage of two ormore molecules by non-covalent or covalent bonds. In some embodiments,where linking of two or more molecules occurs by a covalent bond, thetwo or more molecules can be fused together, or cross-linked together.In some embodiments, where linking of two or more molecules occurs by anon-covalent bond, the two or more molecules can form a complex.

The term “complex” as used herein refers to a collection of two or moremolecules, connected spatially by means other than a covalentinteraction; for example they can be connected by electrostaticinteractions, hydrogen bound or by hydrophobic interactions (i.e., vander Waals forces).

The term “cross-linked” as used herein refers to a covalent bond formedbetween a polymer chain and a second molecule. The term “cross-linkingreagent” refers to an entity or agent which is an intermediate moleculeto catalyze the covalent linkage of a polymer with an entity, e.g.,first affinity molecule or co-stimulatory factor.

As used herein, the term “fused” means that at least one protein orpeptide is physically associated with a second protein or peptide. Insome embodiments, fusion is typically a covalent linkage, however, othertypes of linkages are encompassed in the term “fused” include, forexample, linkage via an electrostatic interaction, or a hydrophobicinteraction and the like. Covalent linkage can encompass linkage as afusion protein or chemically coupled linkage, for example via adisulfide bound formed between two cysteine residues.

As used herein, the term “fusion polypeptide” or “fusion protein” meansa protein created by joining two or more polypeptide sequences together.The fusion polypeptides encompassed in this invention includetranslation products of a chimeric gene construct that joins the DNAsequences encoding one or more antigens, or fragments or mutantsthereof, with the DNA sequence encoding a second polypeptide to form asingle open-reading frame. In other words, a “fusion polypeptide” or“fusion protein” is a recombinant protein of two or more proteins whichare joined by a peptide bond or via several peptides. In someembodiments, the second protein to which the antigens are fused to is acomplementary affinity molecule which is capable of interacting with afirst affinity molecule of the complementary affinity pair.

The terms “polypeptide” and “protein” may be used interchangeably torefer to a polymer of amino acid residues linked by peptide bonds, andfor the purposes of the claimed invention, have a typical minimum lengthof at least 25 amino acids. The term “polypeptide” and “protein” canencompass a multimeric protein, e.g., a protein containing more than onedomain or subunit. The term “peptide” as used herein refers to asequence of peptide bond-linked amino acids containing less than 25amino acids, e.g., between about 4 amino acids and 25 amino acids inlength. Proteins and peptides can be composed of linearly arranged aminoacids linked by peptide bonds, whether produced biologically,recombinantly, or synthetically and whether composed of naturallyoccurring or non-naturally occurring amino acids, are included withinthis definition. Both full-length proteins and fragments thereof greaterthan 25 amino acids are encompassed by the definition of protein. Theterms also include polypeptides that have co-translational (e.g., signalpeptide cleavage) and post-translational modifications of thepolypeptide, such as, for example, disulfide-bond formation,glycosylation, acetylation, phosphorylation, lipidation, proteolyticcleavage (e.g., cleavage by metalloproteases), and the like.Furthermore, as used herein, a “polypeptide” refers to a protein thatincludes modifications, such as deletions, additions, and substitutions(generally conservative in nature as would be known to a person in theart) to the native sequence, as long as the protein maintains thedesired activity. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental, such as throughmutations of hosts that produce the proteins, or errors due to PCRamplification or other recombinant DNA methods.

By “signal sequence” is meant a nucleic acid sequence which, whenoperably linked to a nucleic acid molecule, facilitates secretion of theproduct (e.g., protein or peptide) encoded by the nucleic acid molecule.In some embodiments, the signal sequence is preferably located 5′ to thenucleic acid molecule.

As used herein, the term “N-glycosylated” or “N-glycosylation” refers tothe covalent attachment of a sugar moiety to asparagine residues in apolypeptide. Sugar moieties can include but are not limited to glucose,mannose, and N-acetylglucosamine. Modifications of the glycans are alsoincluded, e.g., siaylation.

An “antigen presenting cell” or “APC” is a cell that expresses the MajorHistocompatibility complex (MHC) molecules and can display foreignantigen complexed with MHC on its surface. Examples of antigenpresenting cells are dendritic cells, macrophages, B-cells, fibroblasts(skin), thymic epithelial cells, thyroid epithelial cells, glial cells(brain), pancreatic beta cells, and vascular endothelial cells.

The term “functional portion” or “functional fragment” as used in thecontext of a “functional portion of an antigen” refers to a portion ofthe antigen or antigen polypeptide that mediates the same effect as thefull antigen moiety, e.g., elicits an immune response in a subject, ormediates an association with other molecule, e.g., comprises at least onepitope.

A “portion” of a target antigen as that term is used herein will be atleast 3 amino acids in length, and can be, for example, at least 6, atleast 8, at least 10, at least 14, at least 16, at least 17, at least18, at least 19, at least 20 or at least 25 amino acids or greater,inclusive.

The terms “Cytotoxic T Lymphocyte” or “CTL” refers to lymphocytes whichinduce death via apoptosis or other mechanisms in targeted cells. CTLsform antigen-specific conjugates with target cells via interaction ofTCRs with processed antigen (Ag) on target cell surfaces, resulting inapoptosis of the targeted cell. Apoptotic bodies are eliminated bymacrophages. The term “CTL response” is used to refer to the primaryimmune response mediated by CTL cells.

The term “cell mediated immunity” or “CMI” as used herein refers to animmune response that does not involve antibodies or complement butrather involves the activation of, for example, macrophages, naturalkiller cells (NK), antigen-specific cytotoxic T-lymphocytes (T-cells),T-helper cells, neutrophils, and the release of various cytokines inresponse to a target antigen. Stated another way, CMI refers to immunecells (such as T cells and other lymphocytes) which bind to the surfaceof other cells that display a target antigen (such as antigen presentingcells (APC)) and trigger a response. The response may involve eitherother lymphocytes and/or any of the other white blood cells (leukocytes)and the release of cytokines. Cellular immunity protects the body by:(i) activating antigen-specific cytotoxic T-lymphocytes (CTLs) that areable to destroy body cells displaying epitopes of foreign antigen ontheir surface, such as virus-infected cells and cells with intracellularbacteria; (2) activating macrophages and NK cells, enabling them todestroy intracellular pathogens; and (3) stimulating cells to secrete avariety of cytokines or chemokines that influence the function of othercells such as T cells, macrophages or neutrophils involved in adaptiveimmune responses and innate immune responses.

The term “immune cell” as used herein refers to any cell which canrelease a cytokine, chemokine or antibody in response to a direct orindirect antigenic stimulation. Included in the term “immune cells”herein are lymphocytes, including natural killer (NK) cells, T-cells(CD4+ and/or CD8+ cells), B-cells, macrophages; leukocytes; dendriticcells; mast cells; monocytes; and any other cell which is capable ofproducing a cytokine or chemokine molecule in response to direct orindirect antigen stimulation. Typically, an immune cell is a lymphocyte,for example a T-cell lymphocyte.

The term “cytokine” as used herein refers to a molecule released from animmune cell in response to stimulation with an antigen. Examples of suchcytokines include, but are not limited to: GM-CSF; IL-1α; IL-1β; IL-2;IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-17A, IL-17F orother members of the IL-17 family, IL-22, IL-23, IFN-α; IFN-β; IFN-γ;MIP-1α; MIP-1β; TGF-β; TNFα, or TNFβ. The term “cytokine” does notinclude antibodies

The term “subject” as used herein refers to any animal in which it isuseful to elicit an immune response. The subject can be a wild,domestic, commercial or companion animal such as a bird or mammal. Thesubject can be a human. Although in one embodiment of the invention itis contemplated that the immunogenic compositions as disclosed hereincan also be suitable for the therapeutic or preventative treatment inhumans, it is also applicable to warm-blooded vertebrates, e.g.,mammals, such as non-human primates, (particularly higher primates),sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat,rabbits, cows, and non-mammals such as chickens, ducks, or turkeys. Inanother embodiment, the subject is a wild animal, for example a birdsuch as for the diagnosis of avian flu. In some embodiments, the subjectis an experimental animal or animal substitute as a disease model. Thesubject may be a subject in need of veterinary treatment, whereeliciting an immune response to an antigen is useful to prevent adisease and/or to control the spread of a disease, for example SIV,STL1, SFV, or in the case of live-stock, hoof and mouth disease, or inthe case of birds Marek's disease or avian influenza, and other suchdiseases.

As used herein, the term “pathogen” refers to an organism or moleculethat causes a disease or disorder in a subject. For example, pathogensinclude but are not limited to viruses, fungi, bacteria, parasites, andother infectious organisms or molecules therefrom, as well astaxonomically related macroscopic organisms within the categories algae,fungi, yeast, protozoa, or the like.

A “cancer cell” refers to a cancerous, pre-cancerous, or transformedcell, either in vivo, ex vivo, or in tissue culture, that hasspontaneous or induced phenotypic changes that do not necessarilyinvolve the uptake of new genetic material. Although transformation canarise from infection with a transforming virus and incorporation of newgenomic nucleic acid, or uptake of exogenous nucleic acid, it can alsoarise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation/cancer is associated with,e.g., morphological changes, immortalization of cells, aberrant growthcontrol, foci formation, anchorage independence, malignancy, loss ofcontact inhibition and density limitation of growth, growth factor orserum independence, tumor specific markers, invasiveness or metastasis,and tumor growth in suitable animal hosts such as nude mice. See, e.g.,Freshney, CULTURE ANIMAL CELLS: MANUAL BASIC TECH. (3rd ed., 1994).

The term “wild type” refers to the naturally-occurring, normalpolynucleotide sequence encoding a protein, or a portion thereof, orprotein sequence, or portion thereof, respectively, as it normallyexists in vivo.

The term “mutant” refers to an organism or cell with any change in itsgenetic material, in particular a change (i.e., deletion, substitution,addition, or alteration) relative to a wild-type polynucleotide sequenceor any change relative to a wild-type protein sequence. The term“variant” may be used interchangeably with “mutant”. Although it isoften assumed that a change in the genetic material results in a changeof the function of the protein, the terms “mutant” and “variant” referto a change in the sequence of a wild-type protein regardless of whetherthat change alters the function of the protein (e.g., increases,decreases, imparts a new function), or whether that change has no effecton the function of the protein (e.g., the mutation or variation issilent).

The term “pharmaceutically acceptable” refers to compounds andcompositions which may be administered to mammals without unduetoxicity. The term “pharmaceutically acceptable carriers” excludestissue culture medium. Exemplary pharmaceutically acceptable saltsinclude but are not limited to mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like, andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. Pharmaceutically acceptable carriers arewell-known in the art.

It will be appreciated that proteins or polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, can be modified in a given polypeptide, eitherby natural processes such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Known modifications which can be present inpolypeptides of the present invention include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa polynucleotide or polynucleotide derivative, covalent attachment of alipid or lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formulation, gamma-carboxylation, glycation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination.

As used herein, the terms “homologous” or “homologues” are usedinterchangeably, and when used to describe a polynucleotide orpolypeptide, indicate that two polynucleotides or polypeptides, ordesignated sequences thereof, when optimally aligned and compared, forexample using BLAST, version 2.2.14 with default parameters for analignment are identical, with appropriate nucleotide insertions ordeletions or amino-acid insertions or deletions, typically in at least70% of the nucleotides of the nucleotides for high homology. For apolypeptide, there should be at least 30% of amino acid identity in thepolypeptide, or at least 50% for higher homology. The term “homolog” or“homologous” as used herein also refers to homology with respect tostructure. Determination of homologs of genes or polypeptides can beeasily ascertained by the skilled artisan. When in the context with adefined percentage, the defined percentage homology means at least thatpercentage of amino acid similarity. For example, 85% homology refers toat least 85% of amino acid similarity.

As used herein, the term “heterologous” reference to nucleic acidsequences, proteins or polypeptides mean that these molecules are notnaturally occurring in that cell. For example, the nucleic acid sequencecoding for a fusion antigen polypeptide described herein that isinserted into a cell, e.g. in the context of a protein expressionvector, is a heterologous nucleic acid sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Where necessary or desired, optimalalignment of sequences for comparison can be conducted by any variety ofapproaches, as these are well-known in the art.

The term “variant” as used herein may refer to a polypeptide or nucleicacid that differs from the naturally occurring polypeptide or nucleicacid by one or more amino acid or nucleic acid deletions, additions,substitutions or side-chain modifications, yet retains one or morespecific functions or biological activities of the naturally occurringmolecule. Amino acid substitutions include alterations in which an aminoacid is replaced with a different naturally-occurring or anon-conventional amino acid residue. Such substitutions may beclassified as “conservative,” in which case an amino acid residuecontained in a polypeptide is replaced with another naturally occurringamino acid of similar character either in relation to polarity, sidechain functionality or size. Substitutions encompassed by variants asdescribed herein may also be “non conservative,” in which an amino acidresidue which is present in a peptide is substituted with an amino acidhaving different properties (e.g., substituting a charged or hydrophobicamino acid with alanine), or alternatively, in which anaturally-occurring amino acid is substituted with a non-conventionalamino acid. Also encompassed within the term “variant,” when used withreference to a polynucleotide or polypeptide, are variations in primary,secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide).

The term “substantially similar,” when used in reference to a variant ofan antigen or a functional derivative of an antigen as compared to theoriginal antigen means that a particular subject sequence varies fromthe sequence of the antigen polypeptide by one or more substitutions,deletions, or additions, but retains at least 50%, or higher, e.g., atleast 60%, 70%, 80%, 90% or more, inclusive, of the function of theantigen to elicit an immune response in a subject. In determiningpolynucleotide sequences, all subject polynucleotide sequences capableof encoding substantially similar amino acid sequences are considered tobe substantially similar to a reference polynucleotide sequence,regardless of differences in codon sequence. A nucleotide sequence is“substantially similar” to a given antigen nucleic acid sequence if: (a)the nucleotide sequence hybridizes to the coding regions of the nativeantigen sequence, or (b) the nucleotide sequence is capable ofhybridization to nucleotide sequence of the native antigen undermoderately stringent conditions and has biological activity similar tothe native antigen protein; or (c) the nucleotide sequences aredegenerate as a result of the genetic code relative to the nucleotidesequences defined in (a) or (b). Substantially similar proteins willtypically be greater than about 80% similar to the correspondingsequence of the native protein.

Variants can include conservative or non-conservative amino acidchanges, as described below. Polynucleotide changes can result in aminoacid substitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. Variants can also includeinsertions, deletions or substitutions of amino acids, includinginsertions and substitutions of amino acids and other molecules) that donot normally occur in the peptide sequence that is the basis of thevariant, for example but not limited to insertion of ornithine which donot normally occur in human proteins. “Conservative amino acidsubstitutions” result from replacing one amino acid with another thathas similar structural and/or chemical properties. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. For example, the following six groups each containamino acids that are conservative substitutions for one another: (1)Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamicacid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine(K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See, e.g.,Creighton, PROTEINS (W.H. Freeman & Co.,1984).

The choice of conservative amino acids may be selected based on thelocation of the amino acid to be substituted in the peptide, for exampleif the amino acid is on the exterior of the peptide and exposed tosolvents, or on the interior and not exposed to solvents. Selection ofsuch conservative amino acid substitutions is within the skill of one ofordinary skill in the art. Accordingly, one can select conservativeamino acid substitutions suitable for amino acids on the exterior of aprotein or peptide (i.e. amino acids exposed to a solvent). Thesesubstitutions include, but are not limited to the following:substitution of Y with F, T with S or K, P with A, E with D or Q, N withD or G, R with K, G with N or A, T with S or K, D with N or E, I with Lor V, F with Y, S with T or A, R with K, G with N or A, K with R, A withS, K or P.

Alternatively, one can also select conservative amino acid substitutionssuitable for amino acids on the interior of a protein or peptide (i.e.,the amino acids are not exposed to a solvent). For example, one can usethe following conservative substitutions: where Y is substituted with F,T with A or S, I with L or V, W with Y, M with L, N with D, G with A, Twith A or S, D with N, I with L or V, F with Y or L, S with A or T and Awith S, G, T or V. In some embodiments, LF polypeptides includingnon-conservative amino acid substitutions are also encompassed withinthe term “variants.” As used herein, the term “non-conservative”substitution refers to substituting an amino acid residue for adifferent amino acid residue that has different chemical properties.Non-limiting examples of non-conservative substitutions include asparticacid (D) being replaced with glycine (G); asparagine (N) being replacedwith lysine (K); and alanine (A) being replaced with arginine (R).

The term “derivative” as used herein refers to proteins or peptideswhich have been chemically modified, for example by ubiquitination,labeling, pegylation (derivatization with polyethylene glycol) oraddition of other molecules. A molecule is also a “derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties can improve themolecule's solubility, absorption, biological half-life, etc. Themoieties can alternatively decrease the toxicity of the molecule, oreliminate or attenuate an undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in REMINGTON'SPHARMACEUTICAL SCIENCES (21st ed., Tory, ed., Lippincott Williams &Wilkins, Baltimore, Md., 2006).

The term “functional” when used in conjunction with “derivative” or“variant” refers to a protein molecule which possesses a biologicalactivity that is substantially similar to a biological activity of theentity or molecule of which it is a derivative or variant.“Substantially similar” in this context means that the biologicalactivity, e.g., antigenicity of a polypeptide, is at least 50% as activeas a reference, e.g., a corresponding wild-type polypeptide, e.g., atleast 60% as active, 70% as active, 80% as active, 90% as active, 95% asactive, 100% as active or even higher (i.e., the variant or derivativehas greater activity than the wild-type), e.g., 110% as active, 120% asactive, or more, inclusive.

The term “recombinant” when used to describe a nucleic acid molecule,means a polynucleotide of genomic, cDNA, viral, semisynthetic, and/orsynthetic origin, which, by virtue of its origin or manipulation, is notassociated with all or a portion of the polynucleotide sequences withwhich it is associated in nature. The term recombinant as used withrespect to a peptide, polypeptide, protein, or recombinant fusionprotein, means a polypeptide produced by expression from a recombinantpolynucleotide. The term recombinant as used with respect to a host cellmeans a host cell into which a recombinant polynucleotide has beenintroduced. Recombinant is also used herein to refer to, with referenceto material (e.g., a cell, a nucleic acid, a protein, or a vector) thatthe material has been modified by the introduction of a heterologousmaterial (e.g., a cell, a nucleic acid, a protein, or a vector).

The term “vectors” refers to a nucleic acid molecule capable oftransporting or mediating expression of a heterologous nucleic acid towhich it has been linked to a host cell; a plasmid is a species of thegenus encompassed by the term “vector.” The term “vector” typicallyrefers to a nucleic acid sequence containing an origin of replicationand other entities necessary for replication and/or maintenance in ahost cell. Vectors capable of directing the expression of genes and/ornucleic acid sequence to which they are operatively linked are referredto herein as “expression vectors”. In general, expression vectors ofutility are often in the form of “plasmids” which refer to circulardouble stranded DNA molecules which, in their vector form are not boundto the chromosome, and typically comprise entities for stable ortransient expression or the encoded DNA. Other expression vectors thatcan be used in the methods as disclosed herein include, but are notlimited to plasmids, episomes, bacterial artificial chromosomes, yeastartificial chromosomes, bacteriophages or viral vectors, and suchvectors can integrate into the host's genome or replicate autonomouslyin the particular cell. A vector can be a DNA or RNA vector. Other formsof expression vectors known by those skilled in the art which serve theequivalent functions can also be used, for example self replicatingextrachromosomal vectors or vectors which integrates into a host genome.Preferred vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked.

The term “reduced” or “reduce” or “decrease” as used herein generallymeans a decrease by a statistically significant amount relative to areference. For avoidance of doubt, “reduced” means statisticallysignificant decrease of at least 10% as compared to a reference level,for example a decrease by at least 20%, at least 30%, at least 40%, atleast t 50%, or least 60%, or least 70%, or least 80%, at least 90% ormore, up to and including a 100% decrease (i.e., absent level ascompared to a reference sample), or any decrease between 10-100% ascompared to a reference level, as that term is defined herein.

The term “low” as used herein generally means lower by a staticallysignificant amount; for the avoidance of doubt, “low” means astatistically significant value at least 10% lower than a referencelevel, for example a value at least 20% lower than a reference level, atleast 30% lower than a reference level, at least 40% lower than areference level, at least 50% lower than a reference level, at least 60%lower than a reference level, at least 70% lower than a reference level,at least 80% lower than a reference level, at least 90% lower than areference level, up to and including 100% lower than a reference level(i.e., absent level as compared to a reference sample).

The terms “increased” or “increase” as used herein generally mean anincrease by a statically significant amount; such as a statisticallysignificant increase of at least 10% as compared to a reference level,including an increase of at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100% or more, inclusive, including, for example at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 10-foldincrease or greater as compared to a reference level, as that term isdefined herein.

The term “high” as used herein generally means a higher by a staticallysignificant amount relative to a reference; such as a statisticallysignificant value at least 10% higher than a reference level, forexample at least 20% higher, at least 30% higher, at least 40% higher,at least 50% higher, at least 60% higher, at least 70% higher, at least80% higher, at least 90% higher, at least 100% higher, inclusive, suchas at least 2-fold higher, at least 3-fold higher, at least 4-foldhigher, at least 5-fold higher, at least 10-fold higher or more, ascompared to a reference level.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The present invention provides for a flexible and versatile compositionthat can be designed and manufactured to elicit a particular, broadspectrum, or variety of antigenic targets. Table 1 provides a simpleexample guide for envisioning the flexibility of MAPS embodiments:

TABLE 1 Versatility of the MAPS platform

Polymers

One component of MAP consists of a “backbone,” typically a polymer. Thepolymer may be antigenic or non-antigenic. It can be made of a widevariety on substances, as described herein, with the caveat that thepolymer serves as a means of presenting the associated antigen(s) to theimmune system in immunogenic fashion. In some embodiments, the polymeris a synthetic polymer. In some embodiments, the polymer is a naturallyoccurring polymer, e.g., a polysaccharide derived or purified frombacterial cells. In some embodiments, the polysaccharide is derived orpurified from eukaryotic cells, e.g., fungi, insect or plant cells. Inyet other embodiments, the polymer is derived from mammalian cells, suchas virus-infected cells or cancer cells. In general, such polymers arewell known in the art and are encompassed for use in the methods andcompositions as disclosed herein.

In some embodiments, a polymer is a polysaccharide selected from any ofthe following, dextran, Vi polysaccharide of Salmonella typhi,pneumococcal capsular polysaccharide, pneumococcal cell wallpolysaccharide (CWPS), meningococcal polysaccharide, Haemophilusinfluenzae type b polysaccharide, or any another polysaccharide ofviral, prokaryotic, or eukaryotic origin.

In some embodiments, the polysaccharide consists of or comprises anantigenic sugar moiety. For example, in some embodiments, apolysaccharide for use in the methods and immunogenic compositions asdisclosed herein is a Vi polysaccharide of Salmonella typhi. The Vicapsular polysaccharide has been developed against bacterial entericinfections, such as typhoid fever. Robbins et al., 150 J. Infect. Dis.436 (1984); Levine et al., 7 Baillieres Clin. Gastroenterol. 501 (1993).Vi is a polymer of α-1→4-galacturonic acid with an N acetyl at positionC-2 and variable O-acetylation at C-3. The virulence of S. typhicorrelates with the expression of this molecule. Sharma et al., 101 PNAS17492 (2004). The Vi polysaccharide vaccine of S. typhi has severaladvantages: Side effects are infrequent and mild, a single dose yieldsconsistent immunogenicity and efficacy. Vi polysaccharide may bereliably standardized by physicochemical methods verified for otherpolysaccharide vaccines, Vi is stable at room temperature and it may beadministered simultaneously with other vaccines without affectingimmunogenicity and tolerability. Azze et al., 21 Vaccine 2758 (2003).

Thus, the Vi polysaccharide of S. typhi may be cross-linked to a firstaffinity molecule as disclosed herein, for attaching at least oneantigen to the polysaccharide. In some embodiments, the antigen can befrom the same or from another organism, such that the resultingimmunogenic composition confers at least some level of immunity againstone pathogen, or two different pathogens: if the antigen confersprotection against pneumococcus, an immunogenic composition where thepolymer scaffold is a Vi polysaccharide can raise an immunogenicresponse against both S. typhi and pneumococci. Other examples includecombining sugars from encapsulated bacteria (such as meningococcus, S.aureus, pneumococcus, Hib, etc.) and tuberculosis antigens, to providean immunogenic composition that raises an immune response against twodifferent pathogens.

Other polysaccharide (PS) moieties that may be used in the presentinvention in alternative to dextran, bacterial cell wall polysaccharides(CWPS), etc., include carbohydrate antigens of cancers.

Further in regard to pneumococcal polysaccharides, the polysaccharidecan be derived from any of the over 93 serotypes of pneumococcus thathave been identified to date, for example, including but not limited toserotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Additional serotypesmay be identified and included in the present immunogenic composition asdescribed herein. More than one pneumococcal polysaccharide can beincluded as the polymer backbone of the present immunogenic compositionsor in a vaccine comprising the present MAPS compositions.

The polysaccharide can also be derived from the invention, theimmunogenic composition comprises N. meningitidis capsularpolysaccharides from at least one, two, three or four of the serogroupsA, C, W, W135, or Y.

A further embodiment comprises the Type 5, Type 8, or any of thepolysaccharides or oligosaccharides of Staphylococcus aureus.

In some embodiments, the polymer is chimeric polymer comprising morethan one type of polymer. For example a polymer of the immunogeniccomposition as disclosed herein can comprise a portion of polymer A, andthe remaining portion of polymer B. There is no limit to the amount ofdifferent types of polymers which can be used in a single MAPS backboneentity. In some embodiments, where the polymer is a branched polymer,the chain polymer can be polymer A, and the branches can be at least 1or at least 2 or at least 3 or more different polymers.

In some embodiments, the polymer is a branched polymer. In someembodiments, the polymer is a single chain polymer.

In some embodiments, the polymer is a polysaccharide comprising at least10 carbohydrate repeating units, or at least 20, or at least 50, or atleast 75, or at least 100, or at least 150, or at least 200, or at least250, or at least 300, or at least 350, or at least 400, or at least 450,or at least 500, or more than 500 repeating units, inclusive.

In one aspect of the invention, the polysaccharide (PS) can have amolecular mass of <500 kDa or >500 kDa. In another aspect of theinvention, the PS has a molecular mass of <70 kDa.

In some embodiments, a polymer is a large molecular weight polymer,e.g., a polymer can be of an average molecular weight of between about425-500 kDa, inclusive, for example, at least 300 kDa, or at least 350kDa, or at least 400 kDa, or at least 425 kDa, or at least 450 kDa, orat least 500 kDa or greater than 500 kDa, inclusive, but typically lessthan 500 kDa.

In some embodiments, a polymer can be a small molecular weight polymer,e.g., a polymer can be of an average molecular weight of between about60kDA to about 90 kDa, for example, at least 50 kDa, or at least 60 kDa,or at least 70 kDa, or at least 80 kDa, or at least 90 kDa, or at least100 kDa, or greater than 100 kDa, inclusive, but generally less thanabout 120 kDa.

In some embodiments, the polymer is harvested and purified from anatural source; and in other embodiments, the polymer is synthetic.Methods to produce synthetic polymers, including syntheticpolysaccharides, are known to persons of ordinary skill and areencompassed in the compositions and methods as disclosed herein.

Just a few of the polysaccharide polymers that can serve as a backbonefor one or more antigens or antigen types are exemplified in Table 2:

TABLE 2 Example polysaccharide polymer MAPS backbone and associatedexample antigens Protein Antigens Number of Antigen Polysaccharideantigens origins Dextran D90 (60-90 KD) two pneumococcus D150 (150 KD)three pneumococcus D270 (270 KD) three pneumococcus D500 (425-575 KD)two; three; pneumococcus six Pneumococcal Serotype 1 one, two,pneumococcus, capsular three, five tuberculosis, polysaccharidestaphylococcus Serotype 3 five pneumococcus, tuberculosis Serotype 5one; two; pneumococcus, three; five tuberculosis Serotype 6B twopneumococcus Serotype 7 three pneumococcus Serotype 14 one; two;pneumococcus, three; five tuberculosis Serotype 19 three pneumococcusPneumococcal cell wall polysaccharide five pneumococcus S. typhi Vipolysaccharide five pneumococcus

Additional polymers that can be used in the immunogenic MAPScompositions described herein include polyethylene glycol-basedpolymers, poly(ortho ester) polymers, polyacryl carriers, PLGA,polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimers, β-amino esterpolymers, polyphosphoester (PPE), liposomes, polymerosomes, nucleicacids, phosphorothioated oligonucleotides, chitosan, silk, polymericmicelles, protein polymers, virus particles, virus-like-particles (VLPs)or other micro-particles. See, e.g., El-Sayed et al., Smart PolymerCarriers for Enhanced Intracellular Delivery of Therapeutic Molecules, 5Exp. Op. Biol. Therapy, 23 (2005). Biocompatible polymers developed fornucleic acid delivery may be adapted for use as a backbone herein. See,e.g., BIOCOMPATIBLE POL. NUCL. ACID. DELIV. (Domb et al., eds., JohnWiley & Sons, Inc. Hoboken, N.J., 2011).

For example, VLPs resemble viruses, but are non-infectious because theydo not contain any viral genetic material. The expression, includingrecombinant expression, of viral structural proteins, such as envelopeor capsid components, can result in the self-assembly of VLPs. VLPs havebeen produced from components of a wide variety of virus familiesincluding Parvoviridae (e.g., adeno-associated virus), Retroviridae(e.g., HIV), and Flaviviridae (e.g., Hepatitis B or C viruses). VLPs canbe produced in a variety of cell culture systems including mammaliancell lines, insect cell lines, yeast, and plant cells. Recombinant VLPsare particularly advantageous because the viral component can be fusedto recombinant antigens as described herein.

Antigens

The immunogenic compositions as disclosed herein can comprise anyantigen that elicits an immune response in a subject. In someembodiments, at least one or more antigens are associated with thepolymer of the composition. In some embodiments, at least 2, or at least3, or at least 5, or at least 10, or at least 15, or at least 20, or atleast 50, or at least 100, or more than 100 antigens can be associatedwith the polymer as disclosed herein. In some embodiments, where theimmunogenic composition comprises more than one antigen, the antigenscan be the same antigen or they can be a variety of different antigensassociated with the polymer. In some embodiments, where the immunogeniccomposition comprises more than one antigen, the antigens can beantigens from the same pathogen or from different pathogens, oralternatively, can be different antigens from the same pathogen, orsimilar antigens from different serotypes of pathogens.

An antigen for use in the immunogenic compositions and methods describedherein can be any antigen, including, but not limited to pathogenicpeptides, toxins, toxoids, subunits thereof, or combinations thereof(e.g., cholera toxin, tetanus toxoid).

In some embodiments, an antigen, which can be fused to the complementaryaffinity molecule, can be any antigen associated with an infectiousdisease, or cancer or immune disease. In some embodiments, an antigencan be an antigen expressed by any of a variety of infectious agents,including virus, bacterium, fungus or parasite.

In some embodiments, an antigen is derived (e.g., obtained) from apathogenic organism. In some embodiments, the antigen is a cancer ortumor antigen, e.g., an antigen derived from a tumor or cancer cell.

In some embodiments, an antigen derived from a pathogenic organism is anantigen associated with an infectious disease; it can be derived fromany of a variety of infectious agents, including virus, bacterium,fungus or parasite.

In some embodiments, a target antigen is any antigen associated with apathology, for example an infectious disease or pathogen, or cancer oran immune disease such as an autoimmune disease. In some embodiments, anantigen can be expressed by any of a variety of infectious agents,including virus, bacterium, fungus or parasite. A target antigen for usein the methods and compositions as disclosed herein can also include,for example, pathogenic peptides, toxins, toxoids, subunits thereof, orcombinations thereof (e.g., cholera toxin, tetanus toxoid).

Non-limiting examples of infectious viruses include: Retroviridae;Picornaviridae (for example, polio viruses, hepatitis A virus;enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (such as strains that cause gastroenteritis); Togaviridae(for example, equine encephalitis viruses, rubella viruses); Flaviridae(for example, dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (forexample, vesicular stomatitis viruses, rabies viruses); Filoviridae (forexample, ebola viruses); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (for example, influenza viruses); Bungaviridae (forexample, Hantaan viruses, bunga viruses, phleboviruses and Nairoviruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g.,reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae(Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae(papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses);Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zostervirus, cytomegalovirus (CMV), Marek's disease virus, herpes viruses);Poxviridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (such as African swine fever virus); and unclassifiedviruses (for example, the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis (thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses). The compositions and methods described herein arecontemplated for use in treating infections with these viral agents.

Examples of fungal infections that may be addressed by inclusion ofantigens in the present embodiments include aspergillosis; thrush(caused by Candida albicans); cryptococcosis (caused by Cryptococcus);and histoplasmosis. Thus, examples of infectious fungi include, but arenot limited to, Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis,Candida albicans. Components of these organisms can be included asantigens in the MAPS described herein.

In one aspect of the invention, an antigen is derived from an infectiousmicrobe such as Bordatella pertussis, Brucella, Enterococci sp.,Neisseria meningitidis, Neisseria gonorrheae, Moraxella, typeable ornontypeable Haemophilus, Pseudomonas, Salmonella, Shigella,Enterobacter, Citrobacter, Klebsiella, E. coli, Helicobacter pylori,Clostridia, Bacteroides, Chlamydiaceae, Vibrio cholera, Mycoplasma,Treponemes, Borelia burgdorferi, Legionella pneumophilia, Mycobacteriasps (such as M. tuberculosis, M. avium, M. intracellulare, M. kansaii,M. gordonae, M. leprae), Staphylococcus aureus, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus anthracis, Corynebacteriumdiphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Leptospira sps., Pasturella multocida,Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis,Treponema pallidium, Treponema pertenue, and Actinomyces israelli. Thecompositions and methods described herein are contemplated for use intreating or preventing infections against these bacterial agents.

Additional parasite pathogens from which antigens can be derivedinclude, for example: Entamoeba histolytica, Plasmodium falciparum,Leishmania sp., Toxoplasma gondii, Rickettsia, and the Helminths.

In another aspect of the invention, an antigen is a truncatedpneumococcal PsaA protein, pneumolysin toxoid pneumococcalserine/threonine protein kinase (StkP), pneumococcal serine/threonineprotein kinase repeating unit (StkPR), pneumococcal PcsB protein,staphylococcal alpha hemolysin, Mycobacterium tuberculosis mtb proteinESAT-6, M. tuberculosis cell wall core antigen, Chlamydia CT144, CT242or CT812 polypeptides or fragments of these, Chlamydia DNA gyrasesubunit B, Chlamydia sulfite synthesis/biphosphate phosphatase,Chlamydia cell division protein FtsY, Chlamydia methionyl-tRNAsynthetase, Chlamydia DNA helicase (uvrD), Chlamydia ATP synthasesubunit I (atpl), or Chlamydia metal dependent hydrolase.

An embodiment of the present invention provides for an immunogeniccomposition targeting the pathogen Myocobacterium tuberculosis (TB), anintracellular bacterial parasite. One example of a TB antigen is TbH9(also known as Mtb 39A). Other TB antigens include, but are not limitedto, DPV (also known as Mtb8.4), 381, Mtb41, Mtb40, Mtb32A, Mtb64, Mtb83,Mtb9.9A, Mtb9.8, Mtb16, Mtb72f, Mtb59f, Mtb88f, Mtb7lf, Mtb46f andMtb31f, wherein “f” indicates that it is a fusion or two or moreproteins.

As noted above, an antigen can be derived from a Chlamydia species foruse in the immunogenic compositions of the present invention.Chlamydiaceae (consisting of Chlamydiae and Chlamydophila), are obligateintracellular gram-negative bacteria. Chlamydia trachomatis infectionsare among the most prevalent bacterial sexually transmitted infections,and perhaps 89 million new cases of genital chlamydial infection occureach year. The Chlamydia of the present invention include, for example,C. trachomatis, Chlamydophila pneumoniae, C. muridarum, C. suis,Chlamydophila abortus, Chlamydophila psittaci, Chlamydophila caviae,Chlamydophila felis, Chlamydophila pecorum, and C. pneumoniae. Animalmodels of chlamydial infection have established that T-cells play acritical role both in the clearance of the initial infection and inprotection from re-infection of susceptible hosts. Hence, theimmunogenic compositions as disclosed herein can be used to provideparticular value by eliciting cellular immune responses againstchlamydial infection.

More specifically, Chlamydial antigens useful in the present inventioninclude DNA gyrase subunit B, sulfite synthesis/biphosphate phosphatase,cell division protein FtsY, methionyl-tRNA synthetase, DNA helicase(uvrD); ATP synthase subunit I (atpl) or a metal-dependent hydrolase(U.S. Patent Application Pub. No. 20090028891). Additional Chlamyidiatrachomatis antigens include CT144 polypeptide, a peptide having aminoacid residues 67-86 of CT144, a peptide having amino acid residues 77-96of CT144, CT242 protein, a peptide having amino acids 109-117 of CT242,a peptide having amino acids 112-120 of CT242 polypeptide, CT812 protein(from the pmpD gene), a peptide having amino acid residues 103-111 ofthe CT812 protein; and several other antigenic peptides from C.trachomatis: NVTQDLTSSTAKLECTQDLI (SEQ ID NO:2), AKLECTQDLIAQGKLIVTNP(SEQ ID NO:3), SNLKRMQKI (SEQ ID NO:4), AALYSTEDL (SEQ ID NO:5),FQEKDADTL (SEQ ID NO:6), QSVNELVYV (SEQ ID NO:7), LEFASCSSL (SEQ IDNO:8), SQAEGQYRL (SEQ ID NO:9), GQSVNELVY (SEQ ID NO:10), and QAVLLLDQI(SEQ ID NO:11). See WO 2009/020553. Additionally, Chlamydia pneumoniaeantigens including homologues of the foregoing polypeptides (see U.S.Pat. No. 6,919,187), can be used an antigens in the immunogeniccompositions and methods as disclosed herein.

Fungal antigens can be derived from Candida species and other yeast; orother fungi (aspergillus, other environmental fungi). Regarding otherparasites, malaria as well as worms and amoebae may provide theantigenic antigen for use in the in the immunogenic compositions andmethods as disclosed herein.

In some embodiments, where the antigen is to generate an anti-influenzaimmunogen, the surface glycoproteins hemagglutinin (HA) andneuraminidase (NA) are generally the antigens of choice. Bothnucleoprotein (NP) polypeptide and matrix (M) are internal viralproteins and therefore not usually considered in vaccine design forantibody-based immunity. Influenza vaccines are used routinely inhumans, and include vaccines derived from inactivated whole influenzavirus, live attenuated influenza virus, or purified and inactivatedmaterials from viral strains. For example, a traditional influenzavaccine can be manufactured using three potentially threatening strainsof flu virus. These strains are usually grown in fertilized chickeneggs, which requires extensive processing including egg inoculation andincubation, egg harvest, virus purification and inactivation, processingand pooling the virus or viral components to the final vaccineformulation, and aseptic filling in the appropriate containers.Typically, this egg-based production cycle takes over 70 weeks. In theevent of a major influenza epidemic, the availability of a potent andsafe vaccine is a major concern. Additionally, there are risksassociated with impurities in eggs, such as antibiotics andcontaminants, that negatively impact vaccine sterility. Moreover,egg-derived flu vaccines are contraindicated for those with severeallergies to egg proteins and people with a history of Guillain-Barrésyndrome. The present invention provides an alternative to the egg-basedinfluenza vaccines, not only be avoiding egg-related selequae, but beproviding a platform for the use of multiple influenza antigens in ahighly controlled platform.

In some embodiments, an antigen for use in the immunogenic compositionsas disclosed herein can also include those used in biological warfare,such as ricin, which may provoke a CMI response.

Additionally, the present invention also provides immunogeniccompositions comprising antigens which raise an immune response againstcancer. In these conjugates, an antigen is an antigen expressed by acancer or tumor, or derived from a tumor. In some embodiments, suchantigens are referred to herein as a “cancer antigen” and are typicallya protein expressed predominantly on the cancer cells, such that theconjugate elicits both potent humoral and potent cellular immunity tothis protein. A large number of cancer-associated antigens have beenidentified, several of which are now being used to make experimentalcancer treatment vaccines and are thus suitable for use in the presentembodiments. Antigens associated with more than one type of cancerinclude Carcinoembryonic antigen (CEA); Cancer/testis antigens, such asNY-ESO-1; Mucin-1 (MUC1) such as Sialyl Tn (STn); Gangliosides, such asGM3 and GD2; p53 protein; and HER2/neu protein (also known as ERBB2).Antigens unique to a specific type of cancer include a mutant form ofthe epidermal growth factor receptor, called EGFRvIII;Melanocyte/melanoma differentiation antigens, such as tyrosinase, MART1,gp100, the lineage related cancer-testis group (MAGE) andtyrosinase-related antigens; Prostate-specific antigen;Leukaemia-associated antigens (LAAs), such as the fusion proteinBCR-ABL, Wilms' tumour protein and proteinase 3; and Idiotype (Id)antibodies. See, e.g., Mitchell, 3 Curr. Opin. Investig. Drugs 150(2002); Dao & Scheinberg, 21 Best Pract. Res. Clin. Haematol. 391(2008).

Another approach in generating an immune response against cancer employsantigens from microbes that cause or contribute to the development ofcancer. These vaccines have been used against cancers includinghepatocellular carcinoma (hepatitis B virus, hepatitis C virus,Opisthorchis viverrin), lymphoma and nasoparyngeal carcinoma(Epstei-Barr virus), colorectal cancer, stomach cancer (Helicobacterpylori), bladder cancer (Schisosoma hematobium), T-cell leukemia (humanT-cell lymphtropic virus), cervical cancer (human papillomavirus), andothers. To date, there have been clinical trials for vaccines targetingBladder Cancer, Brain Tumors, Breast Cancer, Cervical Cancer, KidneyCancer, Melanoma, Multiple Myeloma, Leukemia, Lung Cancer, PancreaticCancer, Prostate Cancer, and Solid Tumors. See Pardoll et al., ABELOFF'SCLIN. ONCOL. (4th ed., Churchill Livingstone, Philadelphia 2008); Sioud,360 Methods Mol. Bio. 277 (2007); Pazdur et al., 30 J. Infusion Nursing30(3):173 (2007); Parmiani et al., 178 J. Immunol. 1975 (2007); Lolliniet al., 24 Trends Immunol. 62 (2003); Schlom et al., 13 Clin. CancerRes. 3776 (2007); Banchereau et al., 392 Nature 245 (1998); Finn, 358New Engl. J. Med. 2704 (2008); Curigliano et al., 7 Exp. Rev. AnticancerTher. 1225 (2007). Marek's Disease virus, a herpes virus that causestumors in poultry, has long been managed by vaccine. Thus, the presentembodiments encompass both preventive or prophylactic anti-cancerimmunogenic compositions and treatment/therapeutic cancer vaccines.

Contemplated proliferative diseases and cancers include AIDS relatedcancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloidleukemia, adenocystic carcinoma, adrenocortical cancer, agnogenicmyeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer,angiosarcoma, astrocytoma, ataxia-telangiectasia, basal cell carcinoma(skin), bladder cancer, bone cancers, bowel cancer, brain and CNStumors, breast cancer, carcinoid tumors, cervical cancer, childhoodbrain tumours, childhood cancer, childhood leukemia, childhood softtissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocyticleukemia, chronic myeloid leukemia, colorectal cancers, cutaneous t-celllymphoma, dermatofibrosarcoma-protuberans,desmoplastic-small-round-cell-tumour, ductal carcinoma, endocrinecancers, endometrial cancer, ependymoma, esophageal cancer, Ewing'ssarcoma, extra-hepatic bile duct cancer, eye cancer, including, e.g.,eye melanoma and retinoblastoma, fallopian tube cancer, fanconi anemia,fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinalcancers, gastrointestinal-carcinoid-tumour, genitourinary cancers, germcell tumors, gestational-trophoblastic disease, glioma, gynecologicalcancers, hematological malignancies, hairy cell leukemia, head and neckcancer, hepatocellular cancer, hereditary breast cancer, Hodgkin'sdisease, human papillomavirus-related cervical cancer, hydatidiformmole, hypopharynx cancer, islet cell cancer, Kaposi's sarcoma, kidneycancer, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumenisyndrome, lip cancer, liposarcoma, lung cancer, lymphedema, lymphoma,non-Hodgkin's lymphoma, male breast cancer,malignant-rhabdoid-tumour-of-kidney, medulloblastoma, melanoma, Merkelcell cancer, mesothelioma, metastatic cancer, mouth cancer, multipleendocrine neoplasia, mycosis fungoides, myelodysplastic syndromes,myeloma, myeloproliferative disorders, nasal cancer, nasopharyngealcancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegenbreakage syndrome, non-melanoma skin cancer,non-small-cell-lung-cancer-(NSCLC), oral cavity cancer, oropharynxcancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasalcancer, parathyroid cancer, parotid gland cancer, penile cancer,peripheral-neuroectodermal-tumours, pituitary cancer, polycythemia vera,prostate cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma,Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, Schwannoma,Sezary syndrome, skin cancer, small cell lung cancer (SCLC), smallintestine cancer, soft tissue sarcoma, spinal cord tumours,squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,testicular cancer, thymus cancer, thyroid cancer,transitional-cell-cancer-(bladder), transitional-cell-cancer(renal-pelvis/ureter), trophoblastic cancer, urethral cancer, urinarysystem cancer, uterine sarcoma, uterus cancer, vaginal cancer, vulvacancer, Waldenstrom's-macroglobulinemia, and Wilms' tumor.

In some embodiments, an antigen for use in the immunogenic compositionsas disclosed herein can include antigens of autoimmune diseases, e.g.,they can be “self-antigens.” Autoimmune diseases contemplated fordiagnosis according to the assays described herein include, but are notlimited to alopecia areata, ankylosing spondylitis, antiphospholipidsyndrome, Addison's disease, aplastic anemia, multiple sclerosis,autoimmune disease of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, Behçet'sDisease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis,chronic fatigue syndrome, chronic inflammatory demyelinating syndrome(CFIDS), chronic inflammatory polyneuropathy, Churg-Strauss syndrome,cicatricial pemphigoid, CREST Syndrome, cold agglutinin disease, Crohn'sdisease, dermatitis herpetiformis, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia, glomerulonephritis, Grave's disease,Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulindependent diabetes (Type I), Lichen Planus, lupus, Meniere's Disease,mixed connective tissue disease, myasthenia gravis, myocarditis,pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma,Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis, temporalarteritis/giant cell arteritis, ulcerative colitis, uveitis, Wegener'ssyndrome, vasculitis and vitiligo. It is generally important to assessthe potential or actual CMI responsiveness in subjects having, orsuspected of having or being susceptible to an autoimmune disease.

In some embodiments, an antigen for use in the immunogenic compositionsas disclosed herein can be an antigen which is associated with aninflammatory disease or condition. Examples of inflammatory diseaseconditions where antigens may be useful include but are not limited toacne, angina, arthritis, aspiration pneumonia, empyema, gastroenteritis,necrotizing enterocolitis, pelvic inflammatory disease, pharyngitis,pleurisy, chronic inflammatory demyelinating polyneuropathy, chronicinflammatory demyelinating polyradiculoneuropathy, and chronicinflammatory demyelinating polyneuropathy, among others.

In some embodiments, an antigen can be an intact (i.e., an entire orwhole) antigen, or a functional portion of an antigen that comprisesmore than one epitope. In some embodiments, an antigen is a peptidefunctional portion of an antigen. By “intact” in this context is meantthat the antigen is the full length antigen as that antigen polypeptideoccurs in nature. This is in direct contrast to delivery of only a smallportion or peptide of the antigen. Delivering an intact antigen to acell enables or facilitates eliciting an immune response to a full rangeof epitopes of the intact antigen, rather than just a single or selectedfew peptide epitopes. Accordingly, the methods and immunogeniccompositions described herein encompass intact antigens associated withthe polymer for a more sensitive and have higher specificity of immuneresponse as compared to use of a single epitope peptide-based antigen.

Alternatively, in some embodiments, an intact antigen can be dividedinto many parts, depending on the size of the initial antigen.Typically, where a whole antigen is a multimer polypeptide, the wholeprotein can be divided into sub-units and/or domains where eachindividual sub-unit or domain of the antigen can be associated with thepolymer according to the methods as disclosed herein. Alternatively, insome embodiments, an intact antigen can be divided into functionalfragments, or parts, of the whole antigen, for example, at least two, orat least 3, or at least 4, or at least 5, or at least 6, or at least 7,or at least 8, or at least 9, or at least 10, or at least 11, or atleast 12, or at least 13, or at least 15, or at least 20, or at least25, or more than 25 portions (e.g., pieces or fragments), inclusive, andwhere each individual functional fragment of the antigen can beassociated with the polymer according to the methods as disclosedherein.

The fragmentation or division of a full length antigen polypeptide canbe an equal division of the full length antigen polypeptide, oralternatively, in some embodiments, the fragmentation is asymmetrical orunequal. As a non-limiting example, where an antigen is divided into twooverlapping fragments, an antigen can be divided into fragments ofapproximately the same (equal) size, or alternatively one fragment canbe about 45% of the whole antigen and the other fragment can be about65%. As further non-limiting examples, a whole antigen can be dividedinto a combination of differently sized fragments, for example, where anantigen is divided into two fragments, fragments can be divided intoabout 40% and about 70%, or about 45% and about 65%; or about 35% andabout 75%; or about 25% and about 85%, inclusive, of the whole antigen.Any combination of overlapping fragments of a full length whole antigenis encompassed for use in the generation of a panel of overlappingpolypeptides of an antigen. As an illustrative example only, where aantigen is divided into 5 portions, the portions can divided equally(i.e., each overlapping fragment is about 21% to 25% of the entire fulllength if the antigen) or unequally (i.e., an antigen can be dividedinto the following five overlapping fragments; fragment 1 is about 25%,fragment 2 is about 5%, fragment 3 is about 35%, fragment 4 is about 10%and fragment 5 is about 25% of the size of the full length antigen,provided each fragment overlaps with at least one other fragment).

Typically, a panel of antigen portions can substantially cover theentire length of the whole (or intact) antigen polypeptide. Accordingly,in some embodiments, an immunogenic composition comprises a polymer withmany different, and/or overlapping fragments of the same intact antigen.Overlapping protein fragments of a antigen can be produced much quickerand cheaper, and with increased stability as compared to the use ofpeptide antigens alone. Further in some embodiments, antigens which arepolypeptides larger than simple peptides are preferred as conformationis important for epitope recognition, and the larger antigenpolypeptides or fragments will provide a benefit over peptide fragments.

One of ordinary skill in the art can divide a whole antigen intooverlapping proteins of an antigen to create a panel of polypeptides ofthe antigen. By way of an illustrative example only, the TB-specificantigen TB1 (CFP also known as culture filtrate-10 or CFP-10) can bedivided into, for example at least seventeen portions to generate apanel of seventeen different polypeptides, each comprising a differentbut overlapping TB-specific antigen TB1 (CFP) fragment. Culture filtrateprotein (CFP-10) (Genbank AAC83445) is a 10 kDa,100 amino acid residueprotein fragment from M. tuberculosis. It is also known as L45 antigenhomologous protein (LHP).

A target antigen for use in the methods and compositions describedherein can be expressed by recombinant means, and can optionally includean affinity or epitope tag to facilitate purification, which methods arewell-known in the art. Chemical synthesis of an oligopeptide, eitherfree or conjugated to carrier proteins, can be used to obtain antigen ofthe invention. Oligopeptides are considered a type of polypeptide. Anantigen can be expressed as a fusion with a complementary affinitymolecule, e.g., but not limited to rhizavidin or a derivative orfunctional fragment thereof. Alternatively, it is also possible toprepare target antigen and then conjugate it to a complementary affinitymolecule, e.g., but not limited to rhizavidin or a derivative orfunctional fragment thereof.

Polypeptides can also by synthesized as branched structures such asthose disclosed in U.S. Pat. Nos. 5,229,490 and 5,390,111. Antigenicpolypeptides include, for example, synthetic or recombinant B-cell andT-cell epitopes, universal T-cell epitopes, and mixed T-cell epitopesfrom one organism or disease and B-cell epitopes from another.

An antigen can obtained through recombinant means or chemicalpolypeptide synthesis, as well as antigen obtained from natural sourcesor extracts, can be purified by means of the antigen's physical andchemical characteristics, such as by fractionation or chromatography.These techniques are well-known in the art.

In some embodiments, an antigen can be solubilized in water, a solventsuch as methanol, or a buffer. Suitable buffers include, but are notlimited to, phosphate buffered saline Ca²⁺/Mg²⁺ free (PBS), normalsaline (150 mM NaCl in water), and Tris buffer. Antigen not soluble inneutral buffer can be solubilized in 10 mM acetic acid and then dilutedto the desired volume with a neutral buffer such as PBS. In the case ofantigen soluble only at acid pH, acetate-PBS at acid pH can be used as adiluent after solubilization in dilute acetic acid. Glycerol can be asuitable non-aqueous solvent for use the compositions, methods and kitsdescribed herein.

Typically, when designing a protein vaccine against a pathogen, anextracellular protein or one exposed to the environment on a virus isoften the ideal candidate as the antigen component in the vaccine.Antibodies generated against that extracellular protein become the firstline of defense against the pathogen during infection. The antibodiesbind to the protein on the pathogen to facilitate antibody opsonizationand mark the pathogen for ingestion and destruction by a phagocyte suchas a macrophage. Antibody opsonization can also kill the pathogen byantibody-dependent cellular cytotoxicity. The antibody triggers arelease of lysis products from cells such as monocytes, neutrophils,eosinophils, and natural killer cells.

In one embodiment of the invention described herein, antigens for use inthe compositions as disclosed herein all wild type proteins, as in theamino acid residues have the sequences found in naturally occurringviruses and have not been altered by selective growth conditions ormolecular biological methods.

In one embodiment, the immunogenic compositions described as herein cancomprise antigens which are glycosylated proteins. In other words, anantigen of interest can each be a glycosylated protein. In oneembodiment of the immunogenic compositions as described herein,antigens, or antigen-fusion polypeptides are O-linked glycosylated. Inanother embodiment of the immunogenic compositions as described herein,antigens, or antigen-fusion polypeptides are N-linked glycosylated. Inyet another embodiment of the immunogenic compositions as describedherein, antigens, or antigen-fusion are both O-linked and N-linkedglycosylated. In other embodiments, other types of glycosylations arepossible, e.g., C-mannosylation. Glycosylation of proteins occurspredominantly in eukaryotic cells. N-glycosylation is important for thefolding of some eukaryotic proteins, providing a co-translational andpost-translational modification mechanism that modulates the structureand function of membrane and secreted proteins. Glycosylation is theenzymatic process that links saccharides to produce glycans, andattaches them to proteins and lipids. In N-glycosylation, glycans areattached to the amide nitrogen of asparagine side chain during proteintranslation. The three major saccharides forming glycans are glucose,mannose, and N-acetylglucosamine molecules. The N-glycosylationconsensus is Asn-Xaa-Ser/Thr, where Xaa can be any of the known aminoacids. O-linked glycosylation occurs at a later stage during proteinprocessing, probably in the Golgi apparatus. In O-linked glycosylation,N-acetyl-galactosamine, O-fucose, O-glucose, and/or N-acetylglucosamineis added to serine or threonine residues. One skilled in the art can usebioinformatics software such as NetNGlyc 1.0 and NetOGlyc Predictionsoftwares from the Technical University of Denmark to find the N- andO-glycosylation sites in a polypeptide in the present invention. TheNetNglyc server predicts N-Glycosylation sites in proteins usingartificial neural networks that examine the sequence context ofAsn-Xaa-Ser/Thr sequons. The NetNGlyc 1.0 and NetOGlyc 3.1 Predictionsoftware can be accessed at the EXPASY website. In one embodiment,N-glycosylation occurs in the target antigen polypeptide of the fusionpolypeptide described herein.

Affinity Molecule Pairs:

As disclosed herein, in some embodiments, an antigen is connected to apolymer via complementary affinity pairs. This connecting of the antigento the polymer is mediated by the polymer being connected to a firstaffinity molecule, which associates a second (e.g., complementary)affinity molecule, which is attached to the antigen. An examplecomplementary affinity pair is biotin/biotin-binding protein.

Exemplary examples of the affinity complementary affinity pairs include,but without limitation, biotin binding proteins or avidin-like proteinsthat bind to biotin. For example, where the first affinity bindingmolecule is biotin (which associates with the polymer), thecomplementary affinity molecule can be a biotin binding protein or anavidin-like protein or a derivative thereof, e.g., but not limited to,avidin, rhizavidin, or streptavidin or variants, derivatives orfunctional portions thereof.

In some embodiments, the first affinity binding molecule is biotin, abiotin derivative, or a biotin mimic, for example, but not limited to,amine-PEG3-biotin (((+)-biotinylation-3-6,9-trixaundecanediamine) or aderivative or functional fragment thereof. A specific biotin mimetic hasa specific peptide motif containing sequence of DX_(a)AX_(b)PX_(c) (SEQID NO:12), or CDX_(a)AX_(b)PX_(c)CG (SEQ ID NO:13), where X_(a) is R orL, X_(b) is S or T, and X_(c) is Y or W. These motifs can bind avidinand Neutravidin, but streptavidin. See, e.g., Gaj et al., 56 Prot.Express. Purif. 54 (2006).

The linkage of the first affinity molecule to the polymer, and thecomplementary affinity molecule to the antigen can be a non-covalentlinkage, or a chemical mechanism, for instance covalent binding,affinity binding, intercalation, coordinate binding and complexation.Covalent binding provides for very stable binding, and is particularlywell-suited for the present embodiments. Covalent binding can beachieved either by direct condensation of existing side chains or by theincorporation of external bridging molecules.

For example, in some embodiments, an antigen can be non-covalentlybonded to one of the pairs in a complementary affixing pair. Inalternative embodiments, an antigen can be covalently bonded or fused toone of the pairs in a complementary affixing pair. Methods forgeneration of fusion proteins are well known in the art, and arediscussed herein.

In other embodiments, a first affinity binding molecule is linked to thepolymer by a non-covalent bond, or by a covalent bond. In someembodiments, a cross-linking reagent is used to covalently bond thefirst affinity binding molecule to the polymer as disclosed herein.

In some embodiments, the first affinity binding molecule associates withthe complementary affinity molecule by non-covalent bond association asknown in the art, including, but not limited to, electrostaticinteraction, hydrogen bound, hydrophobic interaction (i.e., van derWaals forces), hydrophilic interactions, and other non-covalentinteractions. Other higher order interactions with intermediate moietiesare also contemplated.

In some embodiments, the complementary affinity molecule is anavidin-related polypeptide. In specific embodiments, the complementaryaffinity molecule is rhizavidin, such as recombinant rhizavidin. Inparticular, the recombinant rhizavidin is a modified rhizavidin that canbe expressed in E. coli with a high yield. The typical yield is >30 mgper liter of E. coli culture. Rhizavidin has a lower sequence homologyto egg avidin (22.4% sequence identity and 35.0% similarity) comparedwith other avidin-like proteins. Use of the modified rhizavidin reducesthe risk of the MAPS inducing an egg-related allergic reaction in asubject. Moreover, antibody to recombinant modified rhizavidin has noapparent cross-reactivity to egg avidin (and vice versa).

More specifically, some embodiments comprise a modified rhizavidindesigned for recombinant expression in E. coli. The coding sequence forthe rhizavidin gene was optimized using E. coli expression codons, toavoid any difficulty during expression in E. coli due to rare codonspresent in original gene. To simplify the construct, after abioinformatics and structure-based analysis, the first 44 residues offull length rhizavidin were removed, as these were found to beunnecessary for the core structure and function. The correct folding ofrecombinant protein was improved by added an E. coli secretion signalsequence to the N-terminal of the shortened rhizavidin (45-179), tofacilitate the translocation of recombinant protein into the periplasmicspace of E. coli cells where the functionally important disulfide bondin rhizavidin can form correctly. The modified recombinant rhizavidinforms a dimer, compared with known avidin-like proteins which formtetramers, further improving expression of the recombinantrhizavidin-antigen fusion as a soluble protein in E. coli.

Moreover, as discussed in further detail elsewhere herein, to improvethe expression and solubility of fusion antigens in E. coli, a flexiblelinker region was added between rhizavidin and the antigen protein.Additionally, based on the biotinformatics and structural analysis,different antigen constructs were cloned and expressed: either fulllength antigen, or the important functional domain, or chimera proteinswere comprising with two different antigens.

Additional affinity pairs that may be useful in the methods andcompositions described herein include antigen-antibody,metal/ion-metal/ion-binding protein, lipid/lipid binding protein,saccharide/saccharide binding protein, amino acid/peptide/amino acid orpeptide binding protein, enzyme-substrate or enzyme-inhibitor,ligand-agonist/receptor, or biotin mimetic. When using alternativeaffinity pairs, alternative means of attaching the respective polymerand antigen may also be employed, such as in vitro enzymatic reactionsrather than genetic fusion. More specifically, antigen-antibody affinitypair provides for a very strong and specific interaction. The antigencan be any epitope including protein, peptide, nucleic acid, lipid,poly/oligosaccharide, ion, etc. The antibody can be any type ofimmunoglobulin, or the Ag-binding portion of an immunoglobulin, such asa Fab fragment. Regarding metal/ion-metal/ion binding protein, examplesinclude Ni NTA vs. histidine-tagged protein, or Zn vs. Zn bindingprotein. Regarding lipid/lipid binding protein, examples includecholesterol vs. cholesterol binding protein. Regardingsaccharide/saccharide binding protein, examples include maltose vs.maltose binding protein, mannose/glucose/oligosaccharide vs. lectin.Enzyme-substrate/inhibitors include substrates from a wide range ofsubstances, including protein, peptide, amino acid, lipid, sugar, orions. The inhibitor can be the analog of the real substrate which cangenerally bind to the enzymes more tightly and even irreversibly. Forexample, trypsin vs. soy trypsin inhibitor. The inhibitor can be naturalor synthetic molecule. Regarding other ligand/agonist-receptor, ligandcan be from a wide range of substance, including protein, peptide, aminoacid, lipid, sugar, ion, agonist can be the analog of the real ligand.Examples include the LPS vs. TLR4 interaction.

Cross-Linking Reagents:

Many bivalent or polyvalent linking agents are useful in couplingprotein molecules to other molecules. For example, representativecoupling agents can include organic compounds such as thioesters,carbodiimides, succinimide esters, disocyanates, glutaraldehydes,diazobenzenes and hexamethylene diamines. This listing is not intendedto be exhaustive of the various classes of coupling agents known in theart but, rather, is exemplary of the more common coupling agents. SeeKillen & Lindstrom, 133 J. Immunol. 1335 (1984); Jansen et al., 62 Imm.Rev. 185 (1982); Vitetta et al.

In some embodiments, cross-linking reagents agents described in theliterature are encompassed for use in the methods, immunogeniccompositions and kits as disclosed herein. See, e.g., Ramakrishnan, etal., 44 Cancer Res. 201 (1984) (describing the use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester)); Umemoto et al., U.S.Pat. No. 5,030,719 (describing the use of a halogenated acetyl hydrazidederivative coupled to an antibody by way of an oligopeptide linker).Particular linkers include: (a) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (b) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (c) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (d) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G); and (f) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkages or linking agents described above contain components thathave different attributes, thus leading to conjugates with differingphysio-chemical properties. For example, sulfo-NHS esters of alkylcarboxylates are more stable than sulfo-NHS esters of aromaticcarboxylates. NHS-ester containing linkers are less soluble thansulfo-NHS esters. Further, the linker SMPT contains a stericallyhindered disulfide bond, and can form conjugates with increasedstability. Disulfide linkages, are in general, less stable than otherlinkages because the disulfide linkage can be cleaved in vitro,resulting in less conjugate available. Sulfo-NHS, in particular, canenhance the stability of carbodimide couplings. Carbodimide couplings(such as EDC) when used in conjunction with sulfo-NHS, forms esters thatare more resistant to hydrolysis than the carbodimide coupling reactionalone.

Exemplary cross-linking molecules for use in the methods and immunogeniccompostions as disclosed herein include, but are not limited to thoselisted in Tables 3 and 4.

TABLE 3 Exemplary homobifunctional crosslinkers* CrosslinkingCrosslinker Reactive Target Groups, Features Example Products Amine-to-NHS esters DSG; DSS; BS3; TSAT Amine (trifunctional); BioconjugateToolkit Reagent Pairs NHS esters, PEG BS(PEG)5; BS(PEG)9 spacer NHSesters, thiol- DSP; DTSSP cleavable NHS esters, misc- DST; BSOCOES; EGS;cleavable Sulfo-EGS Imidoesters DMA; DMP; DMS Imidoesters, thiol- DTBPcleavable Other DFDNB; THPP (trifunctional); Aldehyde-Activated DextranKit Sulfhydryl-to- Maleimides BMOE; BMB; BMH; TMEA Sulfhydryl(trifunctional) Maleimides, PEG spacer BM(PEG)2; BM(PEG)3 Maleimides,cleavable BMDB; DTME Pyridyldithiols DPDPB (cleavable) Other HBVS(vinylsulfone) Nonselective Aryl azides BASED (thiol-cleavable)*crosslinking reagents that have the same type of reactive group ateither end. Reagents are classified by what chemical groups they crosslink (left column) and their chemical composition (middle column).Products are listed in order of increasing length within each cell.

TABLE 4 Exemplary heterobifunctional crosslinkers* CrosslinkingCrosslinker Reactive Targets Groups, Features Example Products Amine-to-NHS ester/Maleimide AMAS; BMPS; GMBS and Sulfhydryl Sulfo-GMBS; MBS andSulfo-MBS; SMCC and Sulfo-SMCC; EMCS and Sulfo-EMCS; SMPB andSulfo-SMPB; SMPH; LC-SMCC; Sulfo-KMUS NHS ester/Maleimide, SM(PEG)2;SM(PEG)4; PEG spacer SM(PEG)6; SM(PEG)8; SM(PEG)12; SM(PEG)24 NHS ester/SPDP; LC-SPDP and Pyridyldithiol, Sulfo-LC-SPDP; cleavable SMPT;Sulfo-LC-SMPT NHS esters/Haloacetyl SIA; SBAP; SIAB; Sulfo-SIABAmine-to- NHS ester/Aryl Azide NHS-ASA Nonselective ANB-NOS Sulfo-HSABSulfo-NHS-LC-ASA SANPAH and Sulfo- SANPAH NHS ester/Aryl Azide,Sulfo-SFAD; Sulfo- cleavable SAND; Sulfo-SAED NHS ester/Diazirine SDAand Sulfo-SDA; LC-SDA and Sulfo-LC- SDA NHS ester/Diazirine, SDAD andSulfo-SDAD cleavable Amine-to-Carboxyl Carbodiimide DCC; EDCSulfhydryl-to- Pyridyldithiol/Aryl APDP Nonselective AzideSulfhydryl-to- Maleimide/Hydrazide BMPH; EMCH; MPBH; Carbohydrate KMUHPyridyldithiol/ BMPH; EMCH; MPBH; Hydrazide KMUH Carbohydrate-to-Hydrazide/Aryl Azide ABH Nonselective Hydroxyl-to- Isocyanate/MaleimidePMPI Sulfhydryl Amine-to-DNA NHS ester/Psoralen SPB *crosslinkingreagents that have the different reactive groups at either end. Reagentsare classified by what chemical groups they cross link (left column) andtheir chemical composition (middle column). Products are listed in orderof increasing length within each cell.Co-Stimulatory Factor

In some embodiments, the immunogenic composition as disclosed hereincomprises at least one co-stimulatory molecule. In some embodiments, theco-stimulatory factor is cross-linked to the polymer. In someembodiments, the co-stimulatory factor is associated to the polymer by acomplementary affinity pair similar to as an antigen is associated withthe polymer. In some embodiments, where the complementary affinity pairwhich links the co-stimulatory factor to the polymer is the same, or adifferent complementary affinity pair which links the antigen to thepolymer.

In some embodiments, at least one, or at least 2, or at least 3, or atleast 5, or at least 10, or at least 15, or at least 20, or at least 50,or at least 100, or more than about 100, inclusive, co-stimulatoryfactors can be associated with the polymer as disclosed herein. In someembodiments, the co-stimulatory factors can be the same co-stimulatorfactor, or they can be a variety of different co-stimulatory factorsassociated with the polymer.

In some embodiments, the co-stimulator factor is a ligand/agonist ofToll like receptors, e.g., but not limited to TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, etc. In some embodiments, aco-stimulator factor is a NOD ligand/agonist, or an activator/agonist ofthe inflammasome. Without wishing to be bound by theory, theinflammasome is a multiprotein oligomer consisting of caspase 1, PYCARD,NALP and sometimes caspase 5 or caspase 11 and promotes the maturationof inflammatory cytokines interleukin 1β and interleukin 18.

In some embodiments, a co-stimulator factor is a cytokine. In someembodiments, a cytokine is selected from the group consisting of:GM-CSF; IL-1α; IL-1β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10;IL-12; IL-23; IFN-α; IFN-β; IFN-β; IFN-γ; MIP-1α; MIP-1β; TGF-β; TNFα,and TNFβ. In some embodiments, the co-stimulatory factor is an adjuvant,which may be associated with the polymer, as just discussed, or may beadded to the MAPS composition prior to or concurrent with administrationto a subject. Adjuvants are further described elsewhere herein.

Production of Antigens and Antigens Fused to the Complementary AffinityMolecule

Recombinant proteins may be conveniently expressed and purified by aperson skilled in the art, or by using commercially available kits, forexample PROBoND™ Purification System (Invitrogen Corp., Carlsbad,Calif.). In some embodiments, recombinant antigens can be synthesizedand purified by protein purification methods using bacterial expressionsystems, yeast expression systems, baculovirus/insect cell expressionsystem, mammalian cell expression systems, or transgenic plant or animalsystems as known to persons of ordinary skill in the art.

The fusion polypeptides described herein can all be synthesized andpurified by protein and molecular methods that are well known to oneskilled in the art. Molecular biology methods and recombinantheterologous protein expression systems are used. For example,recombinant protein can be expressed in bacteria, mammalian, insect,yeast, or plant cells; or in transgenic plant or animal hosts.

In one embodiment, provided herein is an isolated polynucleotideencoding a fusion polypeptide or a non-fusion polypeptide describedherein. Conventional polymerase chain reaction (PCR) cloning techniquescan be used to construct a chimeric or fusion coding sequence encoding afusion polypeptide as described herein. A coding sequence can be clonedinto a general purpose cloning vector such as pUC19, pBR322,pBLUESCRIPT® vectors (Stratagene, Inc.) or pCR TOPO® (Invitrogen). Theresultant recombinant vector carrying the nucleic acid encoding apolypeptide as described herein can then be used for further molecularbiological manipulations such as site-directed mutagenesis to create avariant fusion polypeptide as described herein or can be subcloned intoprotein expression vectors or viral vectors for protein synthesis in avariety of protein expression systems using host cells selected from thegroup consisting of mammalian cell lines, insect cell lines, yeast,bacteria, and plant cells.

Each PCR primer should have at least 15 nucleotides overlapping with itscorresponding templates at the region to be amplified. The polymeraseused in the PCR amplification should have high fidelity such asPfuULTRA® polymerase (Stratagene) for reducing sequence mistakes duringthe PCR amplification process. For ease of ligating several separate PCRfragments together, for example in the construction of a fusionpolypeptide, and subsequently inserting into a cloning vector, the PCRprimers should also have distinct and unique restriction digestion siteson their flanking ends that do not anneal to the DNA template during PCRamplification. The choice of the restriction digestion sites for eachpair of specific primers should be such that the fusion polypeptidecoding DNA sequence is in-frame and will encode the fusion polypeptidefrom beginning to end with no stop codons. At the same time the chosenrestriction digestion sites should not be found within the coding DNAsequence for the fusion polypeptide. The coding DNA sequence for theintended polypeptide can be ligated into cloning vector pBR322 or one ofits derivatives, for amplification, verification of fidelity andauthenticity of the chimeric coding sequence, substitutions/or specificsite-directed mutagenesis for specific amino acid mutations andsubstitutions in the polypeptide.

Alternatively the coding DNA sequence for the polypeptide can be PCRcloned into a vector using for example, the TOPO® cloning methodcomprising topoisomerase-assisted TA vectors such as pCR®-TOPO,pCR®-Blunt II-TOPO, pENTR/D-TOPO®, and pENTR/SD/D-TOPO®. (Invitrogen,Inc., Carlsbad, Calif.). Both pENTR/D-TOPO®, and pENTR/SD/D-TOPO® aredirectional TOPO entry vectors which allow the cloning of the DNAsequence in the 5′→3′ orientation into a GATEWAY® expression vector.Directional cloning in the 5′→3′ orientation facilitates theunidirectional insertion of the DNA sequence into a protein expressionvector such that the promoter is upstream of the 5′ ATG start codon ofthe fusion polypeptide coding DNA sequence, enabling promoter drivenprotein expression. The recombinant vector carrying the coding DNAsequence for the fusion polypeptide can be transfected into andpropagated in general cloning E. coli such as XL1Blue, SURE®(STRATAGENE®) and TOP-10 cells (Invitrogen).

One skilled in the art would be able to clone and ligate the codingregion of the antigen of interest with the coding region of thecomplementary affinity molecule to construct a chimeric coding sequencefor a fusion polypeptide comprising the antigen or a fragment thereofand the complementary affinity molecule of a derivative thereof usingspecially designed oligonucleotide probes and polymerase chain reaction(PCR) methodologies that are well known in the art. One skilled in theart would also be able to clone and ligate the chimeric coding sequencefor a fusion protein into a selected vector, e.g., bacterial expressionvector, an insect expression vector or baculovirus expression vector.The coding sequences of antigen and the target antigen polypeptide orfragment thereof should be ligated in-frame and the chimeric codingsequence should be ligated downstream of the promoter, and between thepromoter and the transcription terminator. Subsequent to that, therecombinant vector is transfected into regular cloning E. coli, such asXL1Blue. Recombinant E. coli harboring the transfer vector DNA is thenselected by antibiotic resistance to remove any E. coli harboringnon-recombinant plasmid DNA. The selected transformant E. coli are grownand the recombinant vector DNA can be subsequently purified fortransfection into S. frugiperda cells.

In some embodiments, the antigens as disclosed herein can comprise asignal peptide for translocation into periplasmic space of bacteria. Thesignal peptide is also called a leader peptide in the N-terminus, whichmay or may not be cleaved off after the translocation through themembrane. One example of a signal peptide is MKKIWLALAGLVLAFSASA (SEQ IDNO:1) as disclosed herein. Another signal sequence isMAPFEPLASGILLLLWLIAPSRA (SEQ ID NO:14). Other examples of signalpeptides can be found at SPdb, a Signal Peptide Database, which is foundat the world wide web site of “proline.bic.nus.edu.sg/spdb/”.

In some embodiments, where the antigen is fused to a complementaryaffinity protein, the signal sequence can be located at the N-terminalof the complementary affinity protein. For example, if an antigen isfused to an avidin-like protein, the signal sequence can be located atthe N-terminal of the complementary affinity protein. In someembodiments, the signal sequence is cleaved off from the complementaryaffinity protein before the complementary affinity protein associateswith the first affinity molecule.

In some embodiments, an antigen and/or complementary affinity protein asdescribed herein lacks a signal sequence.

The polypeptides described herein can be expressed in a variety ofexpression host cells e.g., bacteria, yeasts, mammalian cells, insectcells, plant cells, algal cells such as Chlamadomonas, or in cell-freeexpression systems. In some embodiments the nucleic acid can besubcloned from the cloning vector into a recombinant expression vectorthat is appropriate for the expression of fusion polypeptide inbacteria, mammalian, insect, yeast, or plant cells or a cell-freeexpression system such as a rabbit reticulocyte expression system. Somevectors are designed to transfer coding nucleic acid for expression inmammalian cells, insect cells and year in one single recombinationreaction. For example, some of the GATEWAY® (Invitrogen) destinationvectors are designed for the construction of baculovirus, adenovirus,adeno-associated virus (AAV), retrovirus, and lentiviruses, which uponinfecting their respective host cells, permit heterologous expression offusion polypeptides in the appropriate host cells. Transferring a geneinto a destination vector is accomplished in just two steps according tomanufacturer's instructions. There are GATEWAY® expression vectors forprotein expression in insect cells, mammalian cells, and yeast.Following transformation and selection in E. coli, the expression vectoris ready to be used for expression in the appropriate host.

Examples of other expression vectors and host cells are the strong CMVpromoter-based pcDNA3.1 (Invitrogen) and pCINEO vectors (Promega) forexpression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat,and MCF-7; replication incompetent adenoviral vector vectors pADENO-X™,pAd5F35, pLP-ADENO™-X-CMV (CLONTECH®), pAd/CMV/V5-DEST, pAd-DEST vector(Invitrogen) for adenovirus-mediated gene transfer and expression inmammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for usewith the RETRO-X™ system from Clontech for retroviral-mediated genetransfer and expression in mammalian cells; pLenti4/V5-DEST™,pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen) forlentivirus-mediated gene transfer and expression in mammalian cells;adenovirus-associated virus expression vectors such as pAAV-MCS,pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) for adeno-associatedvirus-mediated gene transfer and expression in mammalian cells; BACpak6baculovirus (Clontech) and pFASTBAC™ HT (Invitrogen) for the expressionin S. frugiperda 9 (Sf9), Sf11, Tn-368 and BTI-TN-5B4-1 insect celllines; pMT/BiP/V5-His (Invitrogen) for the expression in Drosophilaschneider S2 cells; Pichia expression vectors pPICZα, pPICZ, pFLDα andpFLD (Invitrogen) for expression in P. pastoris and vectors pMETα andpMET for expression in P. methanolica; pYES2/GS and pYD1 (Invitrogen)vectors for expression in yeast S. cerevisiae.

Recent advances in the large scale expression heterologous proteins inChlamydomonas reinhardtii are described. Griesbeck., 34 Mol. Biotechnol.213 (2006); Fuhrmann, 94 Methods Mol Med. 191 (2006). Foreignheterologous coding sequences are inserted into the genome of thenucleus, chloroplast and mitochondria by homologous recombination. Thechloroplast expression vector p64 carrying the most versatilechloroplast selectable marker aminoglycoside adenyl transferase (aadA),which confer resistance to spectinomycin or streptomycin, can be used toexpress foreign protein in the chloroplast. The biolistic gene gunmethod can be used to introduce the vector in the algae. Upon its entryinto chloroplasts, the foreign DNA is released from the gene gunparticles and integrates into the chloroplast genome through homologousrecombination.

Also included in the invention are complementary affinity molecule fusedto an antigen. In some embodiments, the fusion construct can alsooptionally comprise purification tags, and/or secretion signal peptides.These fusion proteins may be produced by any standard method. Forexample, for production of a stable cell line expressing anantigen-complementary affinity molecule fusion protein, PCR-amplifiedantigen nucleic acids may be cloned into the restriction site of aderivative of a mammalian expression vector. For example, KA, which is aderivative of pcDNA3 (Invitrogen) contains a DNA fragment encoding aninfluenza virus hemagglutinin tag (HA). Alternatively, vectorderivatives encoding other tags, such as c-myc or poly Histidine tags,can be used. The antigen-complementary affinity molecule fusionexpression construct may be co-transfected, with a marker plasmid, intoan appropriate mammalian cell line (e.g., COS, HEK293T, or NIH 3T3cells) using, for example, LIPOFECTAMINE™ (Gibco-BRL, Gaithersburg, Md.)according to the manufacturer's instructions, or any other suitabletransfection technique known in the art. Suitable transfection markersinclude, for example, β-galactosidase or green fluorescent protein (GFP)expression plasmids or any plasmid that does not contain the samedetectable marker as the antigen-complementary affinity molecule fusionprotein. The fusion protein expressing cells can be sorted and furthercultured, or the tagged antigen-complementary affinity molecule fusionprotein can be purified. In some embodiments, an antigen-complementaryaffinity molecule fusion protein is amplified with a signal peptide. Inalternative embodiments, a cDNA encoding an antigen-complementaryaffinity molecule fusion protein can be amplified without the signalpeptide and subcloned into a vector (pSecTagHis) having a strongsecretion signal peptide. In another example, antigen-complementaryaffinity molecule fusion protein can have an alkaline phosphatase (AP)tag, or a histadine (His) tag for purification. Any method known topersons of ordinary skill in the art for protein purification of theantigen and/or antigen-complementary affinity molecule fusion protein isencompassed for use in the methods of the invention.

In some embodiments, any of the polypeptides described herein isproduced by expression from a recombinant baculovirus vector. In anotherembodiment, any of the polypeptides described herein is expressed by aninsect cell. In yet another embodiment, any of the polypeptidesdescribed herein is isolated from an insect cell. There are severalbenefits of protein expression with baculovirus in insect cells,including high expression levels, ease of scale-up, production ofproteins with posttranslational modifications, and simplified cellgrowth. Insect cells do not require CO₂ for growth and can be readilyadapted to high-density suspension culture for large-scale expression.Many of the post-translational modification pathways present inmammalian systems are also utilized in insect cells, allowing theproduction of recombinant protein that is antigenically,immunogenically, and functionally similar to the native mammalianprotein.

Baculoviruses are DNA viruses in the family Baculoviridae. These virusesare known to have a narrow host-range that is limited primarily toLepidopteran species of insects (butterflies and moths). The baculovirusAutographa californica Nuclear Polyhedrosis Virus (AcNPV), which hasbecome the prototype baculovirus, replicates efficiently in susceptiblecultured insect cells. AcNPV has a double-stranded closed circular DNAgenome of about 130,000 base-pairs and is well characterized with regardto host range, molecular biology, and genetics. The BaculovirusExpression Vector System (BEVS) is a safe and rapid method for theabundant production of recombinant proteins in insect cells and insects.Baculovirus expression systems are powerful and versatile systems forhigh-level, recombinant protein expression in insect cells. Expressionlevels up to 500 mg/1 have been reported using the baculovirusexpression system, making it an ideal system for high-level expression.Recombinant baculoviruses that express foreign genes are constructed byway of homologous recombination between baculovirus DNA and chimericplasmids containing the gene sequence of interest. Recombinant virusescan be detected by virtue of their distinct plaque morphology andplaque-purified to homogeneity.

Recombinant fusion proteins described herein can be produced in insectcells including, but not limited to, cells derived from the Lepidopteranspecies S. frugiperda. Other insect cells that can be infected bybaculovirus, such as those from the species Bombyx mori, Galleriamellanoma, Trichplusia ni, or Lamanthria dispar, can also be used as asuitable substrate to produce recombinant proteins described herein.Baculovirus expression of recombinant proteins is well known in the art.See U.S. Pat. No. 4,745,051; No. 4,879,236; No. 5,179,007; No.5,516,657; No. 5,571,709; No. 5,759,809. It will be understood by thoseskilled in the art that the expression system is not limited to abaculovirus expression system. What is important is that the expressionsystem directs the N-glycosylation of expressed recombinant proteins.The recombinant proteins described herein can also be expressed in otherexpression systems such as Entomopox viruses (the poxviruses ofinsects), cytoplasmic polyhedrosis viruses (CPV), and transformation ofinsect cells with the recombinant gene or genes constitutive expression.A good number of baculovirus transfer vectors and the correspondingappropriately modified host cells are commercially available, forexample, pAcGP67, pAcSECG2TA, pVL1392, pVL1393, pAcGHLT, and pAcAB4 fromBD Biosciences; pBAC-3, pBAC-6, pBACgus-6, and pBACsurf-1 from NOVAGEN®,and pPolh-FLAG and pPolh-MAT from SIGMA ALDRICH®.

The region between the promoter and the transcriptional terminator canhave multiple restriction enzyme digestion sites for facilitatingcloning of the foreign coding sequence, in this instance, the coding DNAsequence for an antigen polypeptide, and a complementary affinitymolecule. Additional sequences can be included, e.g., signal peptidesand/or tag coding sequences, such as His-tag, MAT-Tag, FLAG tag,recognition sequence for enterokinase, honeybee melittin secretionsignal, beta-galactosidase, glutathione S-transferase (GST) tag upstreamof the MCS for facilitating the secretion, identification, properinsertion, positive selection of recombinant virus, and/or purificationof the recombinant protein.

In some embodiments, the fusion protein can comprise an N-terminalsignal sequence as disclosed herein. In some embodiments, the signalsequence is attached to the N-terminal of the complementary affinitymolecule as disclosed herein.

In some embodiments, a fusion polypeptide as described herein has aspacer peptide, e.g., a 14-residue spacer (GSPGISGGGGGILE) (SEQ IDNO:15) separating antigen from the complementary affinity molecule. Thecoding sequence of such a short spacer can be constructed by annealing acomplementary pair of primers. One of skill in the art can design andsynthesize oligonucleotides that will code for the selected spacer.Spacer peptides should generally have non-polar amino acid residues,such as glycine and proline.

Standard techniques known to those of skill in the art can be used tointroduce mutations (to create amino acid substitutions in an antigenpolypeptide sequence of the fusion polypeptide described herein, e.g.,in the antigen in the nucleotide sequence encoding the fusionpolypeptide described herein, including, for example, site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, the variant fusionpolypeptide has less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions, inclusive, relative to the fusion polypeptidesdescribed herein.

Certain silent or neutral missense mutations can also be made in the DNAcoding sequence that do not change the encoded amino acid sequence orthe capability to promote transmembrane delivery. These types ofmutations are useful to optimize codon usage, or to improve recombinantprotein expression and production.

Specific site-directed mutagenesis of a coding sequence for the fusionpolypeptide in a vector can be used to create specific amino acidmutations and substitutions. Site-directed mutagenesis can be carriedout using, e.g., the QUICKCHANGE® site-directed mutagenesis kit fromStratagene according to the manufacturer's instructions.

In one embodiment, described herein are expression vectors comprisingthe coding DNA sequence for the polypeptides described herein for theexpression and purification of the recombinant polypeptide produced froma protein expression system using host cells selected from, e.g.,bacteria, mammalian, insect, yeast, or plant cells. The expressionvector should have the necessary 5′ upstream and 3′ downstreamregulatory elements such as promoter sequences, ribosome recognition andTATA box, and 3′ UTR AAUAAA transcription termination sequence forefficient gene transcription and translation in its respective hostcell. The expression vector is, preferably, a vector having thetranscription promoter selected from a group consisting of CMV(cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, β-actinpromoter, SV40 (simian virus 40) promoter and muscle creatine kinasepromoter, and the transcription terminator selected from a groupconsisting of SV40 poly(A) and BGH terminator; more preferably, anexpression vector having the early promoter/enhancer sequence ofcytomegalovirus and the adenovirus tripartite leader/intron sequence andcontaining the replication orgin and poly(A) sequence of SV40. Theexpression vector can have additional coding regions, such as thoseencoding, for example, 6X-histidine (SEQ ID NO: 28), V5, thioredoxin,glutathione-S-transferase, c-Myc, VSV-G, HSV, FLAG, maltose bindingpeptide, metal-binding peptide, HA and “secretion” signals (Honeybeemelittin, α-factor, PHO, Bip), which can be incorporated into theexpressed fusion polypeptide. In addition, there can be enzyme digestionsites incorporated after these coding regions to facilitate theirenzymatic removal if they are not needed. These additional nucleic acidsare useful for the detection of fusion polypeptide expression, forprotein purification by affinity chromatography, enhanced solubility ofthe recombinant protein in the host cytoplasm, and/or for secreting theexpressed fusion polypeptide out into the culture media or thespheroplast of the yeast cells. The expression of the fusion polypeptidecan be constitutive in the host cells or it can be induced, e.g., withcopper sulfate, sugars such as galactose, methanol, methylamine,thiamine, tetracycline, infection with baculovirus, and(isopropyl-beta-D-thiogalactopyranoside) IPTG, a stable synthetic analogof lactose.

In another embodiment, the expression vector comprising a polynucleotidedescribed herein is a viral vector, such as adenovirus, adeno-associatedvirus (AAV), retrovirus, and lentivirus vectors, among others.Recombinant viruses provide a versatile system for gene expressionstudies and therapeutic applications.

In some embodiments, the fusion polypeptides described herein areexpressed from viral infection of mammalian cells. The viral vectors canbe, for example, adenovirus, adeno-associated virus (AAV), retrovirus,and lentivirus. A simplified system for generating recombinantadenoviruses is presented by He et al., 95 PNAS 2509 (1998). The gene ofinterest is first cloned into a shuttle vector, e.g., pAdTrack-CMV. Theresultant plasmid is linearized by digesting with restrictionendonuclease PmeI, and subsequently cotransformed into E. coli. BJ5183cells with an adenoviral backbone plasmid, e.g. pADEASY-1 ofStratagene's ADEASY™ Adenoviral Vector System. Recombinant adenovirusvectors are selected for kanamycin resistance, and recombinationconfirmed by restriction endonuclease analyses. Finally, the linearizedrecombinant plasmid is transfected into adenovirus packaging cell lines,for example HEK 293 cells (E1-transformed human embryonic kidney cells)or 911 (E1-transformed human embryonic retinal cells). Fallaux, et al. 7Human Gene Ther. 215 (1996). Recombinant adenovirus are generated withinthe HEK 293 cells.

Recombinant lentivirus has the advantage of delivery and expression offusion polypeptides in dividing and non-dividing mammalian cells. TheHIV-1 based lentivirus can effectively transduce a broader host rangethan the Moloney Leukemia Virus (MoMLV)-based retroviral systems.Preparation of the recombinant lentivirus can be achieved using, forexample, the pLenti4/V5-DEST™, pLenti6/V5-DEST™ or pLenti vectorstogether with VIRAPOWER™ Lentiviral Expression systems from Invitrogen,Inc.

Recombinant adeno-associated virus (rAAV) vectors are applicable to awide range of host cells including many different human and non-humancell lines or tissues. rAAVs are capable of transducing a broad range ofcell types and transduction is not dependent on active host celldivision. High titers, >10⁸ viral particle/ml, are easily obtained inthe supernatant and 10¹¹-10¹² viral particle/ml with furtherconcentration. The transgene is integrated into the host genome soexpression is long term and stable.

Large scale preparation of AAV vectors is made by a three-plasmidcotransfection of a packaging cell line: AAV vector carrying the codingnucleic acid, AAV RC vector containing AAV rep and cap genes, andadenovirus helper plasmid pDF6, into 50×150 mm plates of subconfluent293 cells. Cells are harvested three days after transfection, andviruses are released by three freeze-thaw cycles or by sonication.

AAV vectors can be purified by two different methods depending on theserotype of the vector. AAV2 vector is purified by the single-stepgravity-flow column purification method based on its affinity forheparin. Auricchio et. al., 12 Human Gene Ther. 71 (2001); Summerford &Samulski, 72 J. Virol. 1438 (1998); Summerford & Samulski, 5 Nat. Med.587 (1999). AAV2/1 and AAV2/5 vectors are currently purified by threesequential CsCl gradients.

Without wishing to be bound to theory, when proteins are expressed by acell, including a bacterial cell, the proteins are targeted to aparticular part in the cell or secreted from the cell. Thus, proteintargeting or protein sorting is the mechanism by which a cell transportsproteins to the appropriate positions in the cell or outside of it.Sorting targets can be the inner space of an organelle, any of severalinterior membranes, the cell's outer membrane, or its exterior viasecretion. This delivery process is carried out based on informationcontained in the protein itself. Correct sorting is crucial for thecell; errors can lead to diseases.

With some exceptions, bacteria lack membrane-bound organelles as foundin eukaryotes, but they may assemble proteins onto various types ofinclusions such as gas vesicles and storage granules. Also, depending onthe species of bacteria, bacteria may have a single plasma membrane(Gram-positive bacteria), or both an inner (plasma) membrane and anouter cell wall membrane, with an aqueous space between the two calledthe periplasm (Gram-negative bacteria). Proteins can be secreted intothe environment, according to whether or not there is an outer membrane.The basic mechanism at the plasma membrane is similar to the eukaryoticone. In addition, bacteria may target proteins into or across the outermembrane. Systems for secreting proteins across the bacterial outermembrane may be quite complex and play key roles in pathogenesis. Thesesystems may be described as type I secretion, type II secretion, etc.

In most Gram-positive bacteria, certain proteins are targeted for exportacross the plasma membrane and subsequent covalent attachment to thebacterial cell wall. A specialized enzyme, sortase, cleaves the targetprotein at a characteristic recognition site near the proteinC-terminus, such as an LPXTG motif (SEQ ID NO:16) (where X can be anyamino acid), then transfers the protein onto the cell wall. A systemanalogous to sortase/LPXTG (SEQ ID NO: 16), having the motif PEP-CTERM(SEQ ID NO:17), termed exosortase/PEP-CTERM (SEQ ID NO: 17), is proposedto exist in a broad range of Gram-negative bacteria.

Proteins with appropriate N-terminal targeting signals are synthesizedin the cytoplasm and then directed to a specific protein transportpathway. During, or shortly after its translocation across thecytoplasmic membrane, the protein is processed and folded into itsactive form. Then the translocated protein is either retained at theperiplasmic side of the cell or released into the environment. Since thesignal peptides that target proteins to the membrane are keydeterminants for transport pathway specificity, these signal peptidesare classified according to the transport pathway to which they directproteins. Signal peptide classification is based on the type of signalpeptidase (SPase) that is responsible for the removal of the signalpeptide. The majority of exported proteins are exported from thecytoplasm via the general “Secretory (Sec) pathway”. Most well knownvirulence factors (e.g. exotoxins of Staphylococcus aureus, protectiveantigen of Bacillus anthracis, lysteriolysin O of Listeriamonocytogenes) that are secreted by Gram-positive pathogens have atypical N-terminal signal peptide that would lead them to theSec-pathway. Proteins that are secreted via this pathway aretranslocated across the cytoplasmic membrane in an unfolded state.Subsequent processing and folding of these proteins takes place in thecell wall environment on the trans-side of the membrane. In addition tothe Sec system, some Gram-positive bacteria also contain the Tat-systemthat is able to translocate folded proteins across the membrane.Pathogenic bacteria may contain certain special purpose export systemsthat are specifically involved in the transport of only a few proteins.For example, several gene clusters have been identified in mycobacteriathat encode proteins that are secreted into the environment via specificpathways (ESAT-6) and are important for mycobacterial pathogenesis.Specific ATP-binding cassette (ABC) transporters direct the export andprocessing of small antibacterial peptides called bacteriocins. Genesfor endolysins that are responsible for the onset of bacterial lysis areoften located near genes that encode for holin-like proteins, suggestingthat these holins are responsible for endolysin export to the cell wall.Wooldridge, BACT. SECREILD PROTS: SECRETORY MECHS. & ROLE IN PATHOGEN.(Caister Academic Press, 2009)

In some embodiments, the signal sequence useful in the present inventionis OmpA Signal sequence, however any signal sequence commonly known bypersons of ordinary skill in the art which allows the transport andsecretion of antimicrobial agents outside the bacteriophage infectedcell are encompassed for use in the present invention.

Signal sequence that direct secretion of proteins from bacterial cellsare well known in the art, for example as disclosed in Internationalapplication WO 2005/071088. For example, one can use some of thenon-limited examples of signal peptide shown in Table 5, which can beattached to the amino-terminus or carboxyl terminus of the antimicrobialpeptide (Amp) or antimicrobial polypeptide to be expressed by theantimicrobial-agent engineered bacteriophage, e.g., AMP-engineeredbacteriophage. Attachment can be via fusion or chimera composition withselected antigen or antigen-complementary affinity molecule fusionprotein resulting in the secretion from the bacterium infected with theantimicrobial-agent engineered bacteriophage, e.g. AMP-engineeredbacteriophage.

TABLE 5Example signal peptides to direct secretion of a protein or peptide antigen or antigen-complementary affinity molecule fusion protein of a bacterial cellSecretion Signal Peptide Amino Acid sequence Pathway (NH₂—CO₂₎ GeneGenus/Species secA1 MKKIMLVITLILVSPIAQQTEAK Hly (LLO)Listeria monocytogenes D (SEQ ID NO: 18) MKKKIISAILMSTVILSAAAPLSG Usp45Lactococcus lactis VYADT (SEQ ID NO: 19) MKKRKVLIPLMALSTILVSSTGNPag (protective Bacillus anthracis LEVIQAEV (SEQ ID NO: 20) antigen)secA2 MNMKKATIAATAGIAVTAFAAP Iap (invasion- Listeria monocytogenesTIASAST (SEQ ID NO: 21) associated protein p60) MQKTRKERILEALQEEKKNKKSNamA Imo2691 Listeria monocytogenes KKFKTGATIAGVTAIATSITVPGI (autolysin)EVIVSADE (SEQ ID NO: 22) MKKLKMASCALVAGLMFSGLT *BA_0281Bacillus anthracis PNAFAED (SEQ ID NO: 23) (NLP/P60 family)MAKKFNYKLPSMVALTLVGSA * atl (autolysin) Staphylococcus aureusVTAHQVQAAE (SEQ ID NO: 24) Tat MTDKKSENQTEKTETKENKGM Imo0367Listeria monocytogenes TRREMLKLSAVAGTGIAVGATG LGTILNVVDQVDKALT (SEQ IDNO: 25) MAYDSRFDEWVQKLKEESFQN PhoD (alkaline Bacillus subtillisNTFDRRKFIQGAGKIAGLGLGLT phosphatase) IAQSVGAFG (SEQ ID NO: 26)

The polypeptides as described herein, e.g., antigens orantigen-complementary affinity molecule fusion protein can be expressedand purified by a variety methods known to one skilled in the art, forexample, the fusion polypeptides described herein can be purified fromany suitable expression system. Fusion polypeptides can be purified tosubstantial purity by standard techniques, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others; which arewell-known in the art. See, e.g., Scopes, PROTEIN PURIFICATION:PRINCIPLES & PRACTICE (1982); U.S. Pat. No. 4,673,641.

A number of procedures can be employed when recombinant proteins arepurified. For example, proteins having established molecular adhesionproperties can be reversibly fused to the protein of choice. With theappropriate ligand, the protein can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,the protein of choice can be purified using affinity or immunoaffinitycolumns.

After the protein is expressed in the host cells, the host cells can belysed to liberate the expressed protein for purification. Methods oflysing the various host cells are featured in “Sample Preparation-Toolsfor Protein Research” EMD Bioscience and in the Current Protocols inProtein Sciences (CPPS). An example purification method is affinitychromatography such as metal-ion affinity chromatograph using nickel,cobalt, or zinc affinity resins for histidine-tagged fusionpolypeptides. Methods of purifying histidine-tagged recombinant proteinsare described by Clontech using their TALON® cobalt resin and byNOVAGEN® in their pET system manual, 10th edition. Another preferredpurification strategy is immuno-affinity chromatography, for example,anti-myc antibody conjugated resin can be used to affinity purifymyc-tagged fusion polypeptides. When appropriate protease recognitionsequences are present, fusion polypeptides can be cleaved from thehistidine or myc tag, releasing the fusion polypeptide from the affinityresin while the histidine-tags and myc-tags are left attached to theaffinity resin.

Standard protein separation techniques for purifying recombinant andnaturally occurring proteins are well known in the art, e.g., solubilityfractionation, size exclusion gel filtration, and various columnchromatography.

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the protein of interest. The preferred salt is ammonium sulfate.Ammonium sulfate precipitates proteins by effectively reducing theamount of water in the protein mixture. Proteins then precipitate on thebasis of their solubility. The more hydrophobic a protein is, the morelikely it is to precipitate at lower ammonium sulfate concentrations. Atypical protocol includes adding saturated ammonium sulfate to a proteinsolution so that the resultant ammonium sulfate concentration is between20-30%. This concentration will precipitate the most hydrophobic ofproteins. The precipitate is then discarded (unless the protein ofinterest is hydrophobic) and ammonium sulfate is added to thesupernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Exclusion Filtration

The molecular weight of the protein of choice can be used to isolate itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, AMICON® or MILLIPORE®membranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

The protein of choice can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against recombinant or naturallyoccurring proteins can be conjugated to column matrices and the proteinsimmunopurified. All of these methods are well known in the art. It willbe apparent to one of skill that chromatographic techniques can beperformed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech). For example, an antigenpolypeptide can be purified using a PA63 heptamer affinity column. Singhet al., 269, J. Biol. Chem. 29039 (1994).

In some embodiments, a combination of purification steps comprising, forexample: (a) ion exchange chromatography, (b) hydroxyapatitechromatography, (c) hydrophobic interaction chromatography, and (d) sizeexclusion chromatography can be used to purify the fusion polypeptidesdescribed herein.

Cell-free expression systems are also contemplated. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. Commercially available cell-free expressionsystems include the TNT coupled reticulocyte lysate Systems (Promega)which uses rabbit reticulocyte-based in vitro system.

Formulations of an Immune Composition and Methods of Use

Specific embodiments of the present invention provide for use of theimmunogenic compositions as disclosed herein to elicit an immuneresponse in an animal. More specifically, the compositions elicit bothhumoral and cellular immunity, and in many instance mucosal immunity.Embodiments of the present invention provide at least partial protectionfrom or partial improvement after infection by, in particular,pneumococcus. Pneumococci cause a number of diseases, such asmeningitis, pneumonia, bacteremia, and otitis media. Almost one millionchildren die of pneumococcal diseases worldwide every year. S.pneumoniae have been studied extensively, and at least some of thegenomes sequenced. See, e.g., U.S. Pat. No. 7,141,418. Althoughantibodies to the capsular polysaccharides, which define the knownserotypes, confer serotype-specific protection, other protectivemechanisms of immunity have been described. See Malley et al., 88 J.Mol. Med. 135 (2010). These other protective mechanisms include, but arenot limited to, antibodies to noncapsular antigens and T cell responsesto pneumococcal constituents. The application of protein-polysaccharideconjugate vaccine, PCV7, has reduced diseases significantly. Black etal., 24(S2) Vaccine 79 (2006); Hansen et al., 25 Pediatr. Infect. Dis.J. 779 (2006)). Yet, recent studies have shown that the lack of otherserotypes in PCV7 has resulted in emerging replacement pneumococcalserotypes. Pichichero & Casey, 26(S10) Pediatr. Infect. Dis. J. S12(2007).

Certain pneumococcal antigens common to all serotypes of the specieshave been shown to have immunoprotective potential despite theencapsulation, e.g., the surface proteins PspA, PspC, PsaA and thecytotoxin pneumolysin or pneumolysoid mutants (Basset et al., 75 Infect.Immun. 5460 (2007); Briles et al., 18 Vaccine 1707 (2000)); the use ofgenomics and mutational libraries has identified several dozenadditional species-common proteins (Hava & Camilli, 45 Mol. Microbiol.1389 (2002); Wizemann et al., 60 Infect. Immun. 1593 (2001)). Immunityhas been induced by individual antigens in animal models (Alexander etal., 62 Infect. Immun. 5683 (1994); Balachandran et al., 70 Infect.Immun. 2526 (2002); Chung et al., 170 J. Immunol. 1958 (2003); Glover etal., 76 Infect. Immun. 2767 (2008); Wu et al., 175 J. Infect. Dis. 839(1997)), but no vaccine based on a common antigen has been approved forhuman use to date.

In one embodiment, provided herein is a method of vaccinating a mammalcomprising administering the immunogenic composition comprising at leastone, or multiple antigens attached to at least one type of polymerscaffold, e.g., a polysaccharide or carbohydrate polymer for use ineliciting an immune response to the one or more antigens attached to thepolymer when administered to a subject. In some embodiments, the immuneresponse is a humoral and/or cellular immune response.

Accordingly, one aspect of the present invention relates to methods toelicit an immune response in a subject, comprising administering to thesubject an immunogenic composition comprising at least one type of thepolymer, e.g., a polysaccharide, at least one antigen, and at least onecomplementary affinity-molecule pair comprising (i) a first affinitymolecule which associates with the polymer, e.g., a polysaccharide, and(ii) a complementary affinity molecule which associates with theantigen, to attach the antigen to the polymer, e.g., a polysaccharide,(e.g., the first affinity molecule associates with the complementaryaffinity molecule to link the antigen to the polymer, e.g.,polysaccharide).

Accordingly, one aspect of the present invention relates to methods toelicit a humoral and/or cellular immunity to multiple antigens at thesame time, e.g., where the immunogenic composition administered to thesubject comprises a polymer comprising at least 1, or at least 2, or amore, e.g., a plurality of the same or different antigens.

One aspect of the present invention relates to a method of immunizationor vaccinating a subject, e.g., a bird or a mammal, e.g., a humanagainst a pathogen comprises administering an immune composition asdisclosed herein comprising at least one antigen derived from one ormore pathogens. In some embodiments, a subject can be immunized againstat least 1, or at least 2, or at least 2, or at least 3, or at least 5,or at least 10, or at least 15, or at least about 20, or at least 50, orat least about 100, or more than 100 different pathogens at the sametime, where the polymer of the immunogenic composition as thecorresponding different antigens attached.

In some embodiments, a subject can be administered several differentimmunogenic compositions as disclosed herein, for example, a subject canbe administered a composition comprising a polymer with an antigen, or aplurality of antigens, e.g., antigens A, B, C, and D etc., and alsoadministered a composition comprising a polymer comprising a differentantigen, or a different set of antigens, e.g., antigens W, X, Y, and Zetc. Alternatively, a subject can be administered a compositioncomprising a polymer A with an antigen, or a plurality of antigens,e.g., antigens A, B, C, and D, etc., and also administered a compositioncomprising a polymer B comprising the same e.g., antigens A, B, C, and Detc., or a different set of antigens. It is envisioned that the presentinvention provides a methods for the immunization of a subject with asmany antigens as desired, e.g., with a variety of different immunogeniccomplexes as described herein, to enable immunization with as many as100 or more antigens.

In one embodiment, the immunogenic compositions as described hereincomprise a pharmaceutically acceptable carrier. In another embodiment,the immunogenic composition composition described herein is formulatedfor administering to a bird, mammal, or human, as or in a vaccine.Suitable formulations can be found in, for example, Remington'sPharmaceutical Sciences (2006), or Introduction to Pharmaceutical DosageForms (4th ed., Lea & Febiger, Philadelphia, 1985).

In one embodiment, the immunogenic compositions as described hereincomprise pharmaceutically acceptable carriers that are inherentlynontoxic and nontherapeutic. Examples of such carriers include ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and polyethyleneglycol. For all administrations, conventional depot forms are suitablyused. Such forms include, for example, microcapsules, nano-capsules,liposomes, plasters, inhalation forms, nose sprays, sublingual tablets,and sustained release preparations. For examples of sustained releasecompositions, see U.S. Pat. Nos. 3,773,919, 3,887,699, EP 58,481A, EP158,277A, Canadian Patent No. 1176565; Sidman et al., 22 Biopolymers 547(1983); Langer et al., 12 Chem. Tech. 98 (1982). The proteins willusually be formulated at a concentration of about 0.1 mg/ml to 100 mg/mlper application per patient.

In one embodiment, other ingredients can be added to vaccineformulations, including antioxidants, e.g., ascorbic acid; low molecularweight (less than about ten residues) polypeptides, e.g., polyarginineor tripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose, or dextrins; chelatingagents such as EDTA; and sugar alcohols such as mannitol or sorbitol.

In some embodiments, the present MAPS immunogen compositions areadministered with at least one adjuvant. Adjuvants are a heterogeneousgroup of substances that enhance the immunological response against anantigen that is administered simultaneously. In some instances,adjuvants improve the immune response so that less vaccine is needed.Adjuvants serve to bring the antigen—the substance that stimulates thespecific protective immune response—into contact with the immune systemand influence the type of immunity produced, as well as the quality ofthe immune response (magnitude or duration). Adjuvants can also decreasethe toxicity of certain antigens; and provide solubility to some vaccinecomponents. Almost all adjuvants used today for enhancement of theimmune response against antigens are particles or form particlestogether with the antigen. In the book VACCINE DESIGN—SUBUNIT & ADJUVANTAPPROACH (Powell & Newman, Eds., Plenum Press, 1995), many knownadjuvants are described both regarding their immunological activity andregarding their chemical characteristics. The type of adjuvants that donot form particles are a group of substances that act as immunologicalsignal substances and that under normal conditions consist of thesubstances that are formed by the immune system as a consequence of theimmunological activation after administration of particulate adjuvantsystems.

Adjuvants for immunogenic compositions and vaccines are well known inthe art. Examples include, but not limited to, monoglycerides and fattyacids (e.g. a mixture of mono-olein, oleic acid, and soybean oil);mineral salts, e.g., aluminium hydroxide and aluminium or calciumphosphate gels; oil emulsions and surfactant based formulations, e.g.,MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21(purified saponin), AS02 [SBAS2] (oil-in-water emulsion+MPL+QS-21),MPL-SE, Montanide ISA-51 and ISA-720 (stabilised water-in-oil emulsion);particulate adjuvants, e.g., virosomes (unilamellar liposomal vehiclesincorporating influenza haemagglutinin), ASO4 ([SBAS4] Al salt withMPL), ISCOMS (structured complex of saponins and lipids), polylactideco-glycolide (PLG); microbial derivatives (natural and synthetic), e.g.,monophosphoryl lipid A (MPL), Detox (MPL+M. Phlei cell wall skeleton),AGP [RC-529] (synthetic acylated monosaccharide), Detox-PC, DC_Chol(lipoidal immunostimulators able to self-organize into liposomes),OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotidescontaining immunostimulatory CpG motifs), or other DNA structures,modified LT and CT (genetically modified bacterial toxins to providenon-toxic adjuvant effects); endogenous human immunomodulators, e.g.,hGM-CSF or hIL-12 (cytokines that can be administered either as proteinor plasmid encoded), Immudaptin (C3d tandem array), MoGM-CSF,TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I,GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59and inert vehicles, such as gold particles. Additional adjuvants areknown in the art, see, e.g., U.S. Pat. No. 6,890,540; U. S. Patent Pub.No. 2005; 0244420; PCT/SE97/01003.

In some embodiments an adjuvant is a particulate and can have acharacteristic of being slowly biodegradable. Care must be taken toensure that that the adjuvant do not form toxic metabolites. Preferably,in some embodiments, such adjuvants can be matrices used are mainlysubstances originating from a body. These include lactic acid polymers,poly-amino acids (proteins), carbohydrates, lipids and biocompatiblepolymers with low toxicity. Combinations of these groups of substancesoriginating from a body or combinations of substances originating from abody and biocompatible polymers can also be used. Lipids are thepreferred substances since they display structures that make thembiodegradable as well as the fact that they are a critical element inall biological membranes.

In one embodiment, the immunogenic compositions as described herein foradministration must be sterile for administration to a subject.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes), or by gammairradiation.

In some embodiments, the immunogenic compositions described hereinfurther comprise pharmaceutical excipients including, but not limited tobiocompatible oils, physiological saline solutions, preservatives,carbohydrate, protein, amino acids, osmotic pressure controlling agents,carrier gases, pH-controlling agents, organic solvents, hydrophobicagents, enzyme inhibitors, water absorbing polymers, surfactants,absorption promoters and anti-oxidative agents. Representative examplesof carbohydrates include soluble sugars such as hydropropyl cellulose,carboxymethyl cellulose, sodium carboxyl cellulose, hyaluronic acid,chitosan, alginate, glucose, xylose, galactose, fructose, maltose,saccharose, dextran, chondroitin sulfate, etc. Representative examplesof proteins include albumin, gelatin, etc. Representative examples ofamino acids include glycine, alanine, glutamic acid, arginine, lysine,and their salts. Such pharmaceutical excipients are well-known in theart.

In some embodiments, the immunogenic MAPS composition is administered incombination with other therapeutic ingredients including, e.g.,γ-interferon, cytokines, chemotherapeutic agents, or anti-inflammatory,or anti-viral agents. In some embodiments, the immunogenic compositionas disclosed herein can be administered with one or more co-stimulatorymolecules and/or adjuvants as disclosed herein.

In some embodiments, the immunogenic composition is administered in apure or substantially pure form, but may be administered as apharmaceutical composition, formulation or preparation. Such formulationcomprises MAPS described herein together with one or morepharmaceutically acceptable carriers and optionally other therapeuticingredients. Other therapeutic ingredients include compounds thatenhance antigen presentation, e.g., gamma interferon, cytokines,chemotherapeutic agents, or anti-inflammatory agents. The formulationscan conveniently be presented in unit dosage form and may be prepared bymethods well known in the pharmaceutical art. For example, Plotkin andMortimer, in VACCINES (2nd ed., W.B. Saunders Co., 1994) describesvaccination of animals or humans to induce an immune response specificfor particular pathogens, as well as methods of preparing antigen,determining a suitable dose of antigen, and assaying for induction of animmune response.

Formulations suitable for intravenous, intramuscular, intranasal, oral,sublingual, vaginal, rectal, subcutaneous, or intraperitonealadministration conveniently comprise sterile aqueous solutions of theactive ingredient with solutions which are preferably isotonic with theblood of the recipient. Such formulations may be conveniently preparedby dissolving solid active ingredient in water containingphysiologically compatible substances such as sodium chloride (e.g.,0.1M-2.0 M), glycine, and the like, and having a buffered pH compatiblewith physiological conditions to produce an aqueous solution, andrendering the solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Formulations for an intranasal delivery are described in U.S. Pat. Nos.5,427,782; 5,843,451; 6,398,774.

The formulations of the immunogenic compositions can incorporate astabilizer. Illustrative stabilizers are polyethylene glycol, proteins,saccharide, amino acids, inorganic acids, and organic acids which may beused either on their own or as admixtures. Two or more stabilizers maybe used in aqueous solutions at the appropriate concentration and/or pH.The specific osmotic pressure in such aqueous solution is generally inthe range of 0.1-3.0 osmoses, preferably in the range of 0.80-1.2. ThepH of the aqueous solution is adjusted to be within the range of pH5.0-9.0, preferably within the range of pH 6-8.

When oral preparations are desired, the immunogenic compositions can becombined with typical carriers, such as lactose, sucrose, starch, talcmagnesium stearate, crystalline cellulose, methyl cellulose,carboxymethyl cellulose, glycerin, sodium alginate or gum arabic amongothers.

In some embodiments, the immunogenic compositions as described hereincan be administered intravenously, intranasally, intramuscularly,subcutaneously, intraperitoneally, sublingually, vaginal, rectal ororally. In some embodiments, the route of administration is oral,intranasal, subcutaneous, or intramuscular. In some embodiments, theroute of administration is intranasal administration.

Vaccination can be conducted by conventional methods. For example, animmunogenic compositions can be used in a suitable diluent such assaline or water, or complete or incomplete adjuvants. The immunogeniccomposition can be administered by any route appropriate for elicitingan immune response. The immunogenic composition can be administered onceor at periodic intervals until an immune response is elicited. Immuneresponses can be detected by a variety of methods known to those skilledin the art, including but not limited to, antibody production,cytotoxicity assay, proliferation assay and cytokine release assays. Forexample, samples of blood can be drawn from the immunized mammal, andanalyzed for the presence of antibodies against the antigens of theimmunogenic composition by ELISA (see de Boer et. al., 115 Arch Virol.147 (1990) and the titer of these antibodies can be determined bymethods known in the art.

The precise dose to be employed in the formulation will also depend onthe route of administration and should be decided according to thejudgment of the practitioner and each patient's circumstances. Forexample, a range of 25 μg-900 μg total protein can be administeredmonthly for three months.

Ultimately, the attending physician will decide the amount ofimmunogenic composition or vaccine composition to administer toparticular individuals. As with all immunogenic compositions orvaccines, the immunologically effective amounts of the immunogens mustbe determined empirically. Factors to be considered include theimmunogenicity, whether or not the immunogen will be complexed with orcovalently attached to an adjuvant or carrier protein or other carrier,routes of administrations and the number of immunizing dosages to beadministered. Such factors are known in the vaccine art and it is wellwithin the skill of immunologists to make such determinations withoutundue experimentation.

Kits

The present invention also provides for kits for producing animmunogenic composition as disclosed herein which is useful for aninvestigator to tailor an immunogenic composition with their preferredantigens, e.g., for research purposes to assess the effect of anantigen, or a combination of antigens on immune response. Such kits canbe prepared from readily available materials and reagents. For example,such kits can comprise any one or more of the following materials: acontainer comprising a polymer, e.g., a polysaccharide, cross-linkedwith a plurality of first affinity molecules; and a container comprisinga complementary affinity molecule which associates with the firstaffinity molecule, wherein the complementary affinity moleculeassociates with an antigen.

In another embodiment, the kit can comprise a container comprising apolymer, e.g., a polysaccharide, a container comprising a plurality offirst affinity molecules, and a container comprising a cross-linkingreagent for cross-linking the first affinity molecules to the polymer.

In some embodiments, the kit further comprises a means to attach thecomplementary affinity molecule to the antigen, where the means can beby a cross-linking reagent or by some intermediary fusion protein. Insome embodiments, the kit can comprise at least one co-stimulationfactor which can be added to the polymer. In some embodiments, the kitcomprises a cross-linking reagent, for example, but not limited to, CDAP(1-cyano-4-dimethylaminopyridinium tetrafluoroborate), EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sodiumcyanoborohydride; cyanogen bromide; ammonium bicarbonate/iodoacetic acidfor linking the co-factor to the polymer.

A variety of kits and components can be prepared for use in the methodsdescribed herein, depending upon the intended use of the kit, theparticular target antigen and the needs of the user.

In one embodiment, an immunogenic composition or vaccine composition asdescribed herein, when administered to mice, can provoke an immuneresponse that prevents a disease symptom in at least 20% of animalschallenged with 5 LD₅₀ of the immunogenic composition comprisingantigens to which the disease symptom is prevented. Methods ofvaccination and challenging an immunized animal are known to one skilledin the art. For example, a 10 μg aliquot of an immunogenic compositionor vaccine composition as disclosed herein can be prepared in 100 μl PBSand/or with addition of incomplete Freund's adjuvant and injectedintramuscularly per mouse per vaccination. Alternatively, parenteral,intraperitoneal and footpad injections can be used. Volumes of footpadinjections are reduced to 50 μl. Mice can be immunized with animmunogenic composition or vaccine composition as disclosed herein onthree separate occasions with several days, e.g., 14 days interval inbetween.

Efficacy of vaccination can be tested by challenge with the pathogen.Seven days after the last dose of an immunogenic composition, theimmunized mice are challenged intranasally with a pathogenic organismfrom which the antigen was derived. Ether anaesthetized mice (10 g to 12g) can be infected intranasally with 50 μl of PBS-diluted allantoicfluid containing 5 LD₅₀ of the pathogenic organism. Protection can bemeasured by monitoring animal survival and body weight, which isassessed throughout an observation period of 21 days. Severely affectedmice are euthanized. One LD₅₀ of A/Mallard/Pennsylvania/10218/84 isequal to 100-1000 the Tissue Culture Infectious Dose50 (TCID50) assay.

In other embodiments, the immunized mice can be challenged with avariety of different pathogenic organisms, e.g., different pathogenicorganisms from which each of the antigens attached to the polymer arederived. For example, of an immunogenic composition comprises fivedifferent antigens attached to the polymer, e.g., polysaccharide, whereeach antigen is derived from five different pathogenic organisms, theimmunized mice can be challenged with each of the five differentpathogenic organisms, either sequentially (in any order) orconcurrently. One skilled in the art would be able to determine the LD₅₀for each pathogenic organism used to challenge the immunized mice bymethods known in the art. See, e.g., LaBarre & Lowy, 96 J. Virol. Meths.107 (2001); Golub, 59J. Immunol. 7 (1948).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity of understandingit will be readily apparent to one of ordinary skill in the art in lightof the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims. The following is meant to be illustrativeof the present invention; however, the practice of the invention is notlimited or restricted in any way by the examples.

EXAMPLES

The examples presented herein relate to methods to generate animmunogenic complex as described herein and methods and compositionsthereof. In particular, the examples relate to methods to produce amultiple antigen presentation (MAP) complex as disclosed herein, andmethods of use to generate an immune response in a subject.

Example 1 Construction of Recombinant Rhizavidin and Rhizavidin-AntigenFusion Proteins

The recombinant Rhizavidin (rRhavi) used in these studies is anN-terminal modified version that contains only the residues 45 to 179 ofthe wild type protein. To optimize the expression level of rRhavi in E.coli, the gene sequence that encodes Rhizavidin polypeptides (45-179)was re-designed by using E. coli-preferred expression codons, thensynthesized and cloned into the PET21b vector. To facilitate the correctfolding and obtain a high yield of soluble recombinant protein, a DNAsequence encoding an E. coli periplasmic localization signal sequence(19 amino acids, MKKIWLALAGLVLAFSASA, SEQ ID NO:1) was introduced at the5′ end of the synthetic gene of rRhavi. This signal sequence ispredicted to be deleted automatically from the recombinant protein afterits targeting to the periplasm of E. coli during the process ofexpression.

To construct a Rhizavidin-antigen fusion protein, a DNA sequenceencoding a flexible linker region consisting of seven amino acids(GGGGSSS, SEQ ID NO:27) was directly inserted into the 3′ end of thesynthetic rRhavi gene, to help stablize the fusion protein. The genesencoding candidate antigens (full length or desired fragment) wereamplified from the genomic DNA of interested pathogens by routine PCRprocedures and inserted into the rRhavi expression vector just beyondthe linker region.

For protein expression, the plasmids containing target constructs weretransformed into E. coli strain BL21 (DE3) using standard heat-shockprocedure. A single colony was picked freshly from the plate (or aglycerol stock was used later) and inoculated into 30 ml Luria-Bertani(LB) medium containing Ampicillin (Amp+) for an overnight culture at 37°C. On day 2, a 5 ml starting culture was inoculated into 1 liter of LBmedium/Amp+ and grown at 37° C. until OD₆₀₀=1 was reached. After coolingthe medium to 16° C., 0.2 mM final concentration of IPTG was added intothe cultures for an overnight induction.

Proteins were purified from the periplasmic fraction using a modifiedosmotic shock protocol. Briefly, the bacterial cells from the 6 literculture were collected and resuspended in 120 ml buffer containing 30 mMTris (pH 8.0), 20% sucrose and 1 mM EDTA. After stiffing at roomtemperature for 20 mM, the cells were re-pelleted by centrifugation at10,000 rpm for 10 mM. The supernatant was collected as fraction 1, andthe cells were resuspended in 80 ml ice cold solution containing 5 mMMgCl₂, proteinase inhibitor and DNase. After stiffing at 4° C. for 20mM, the mixture was subjected to centrifugation at 13,000 rpm for 20 mMand the supernatant was collected as fraction 2. After adding a finalconcentration of 150 mM NaCl, 10 mM MgCl₂ and 10 mM Imidazole, thesupernatant combining fraction 1 and fraction 2 was applied onto aNi-NTA column. The proteins eluted from the Ni-NTA column were furtherpurified by gel-filtration using superdex 200 column running on AKTApurifier. The peak fractions containing target protein were pooled andconcentrated. The protein concentration was measured by using BCAprotein assay kit from Bio-Rad. Purified proteins were aliquoted,flash-frozen in liquid nitrogen and kept at −80° C. for future use.

Example 2 Biotinylation of Polysaccharide

The biotinylation of polysaccharides was done by using1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as theactivation reagent. Briefly, the polysaccharides were dissolved inLPS-free water at 10 mg/ml (or other concentration as indicated). Att=0, a volume of CDAP (freshly made at 100 mg/ml in acetonitrile) wasslowly added to the polysaccharide solution at a ratio of 1-2 mg CDAP/mgpolysaccharide, while vortexing. Thirty seconds later, a volume of 0.2 Mtriethylamine (TEA) was added (equal or double to the volume of CDAP,depending on the different types of polysaccharide) to raise the pH. At2.5 mM, a volume of biotin derivative (EZ-Link Amine-PEG3-Biotin fromPierce, solubilized at 20 mg/ml in LPS-free water) was added to a finalratio of 1-1.5 mg biotin/mg polysaccharide for an overnight coupling at4° C. (or 1-3 hr coupling at 25° C.). On day 2, 50 mM finalconcentration of glycine or serine was added to terminate the reactionand then the mixture was desalted by passage over a column or dialyzedagainst a large volume of PBS to remove free biotin derivatives. Thebiotin content in the biotinylated polysaccharide was measured by usingthe biotin quantification kit from Pierce and the polysaccharideconcentration was determined by the anthrone assay.

Example 3 Assembly and Purification of MAPS

To assemble a MAPS complex, a volume of biotinylated polysaccharide wasmixed with the candidate rRhavi-antigen fusion proteins in a desiredratio and then incubated at 4° C. or 25° C. overnight. After incubation,the mixture was centrifuged at 13,200 rpm for 3 min to remove theinsoluble aggregates. The supernatant was applied to the gel-filtrationchromatography, using superpose-6 or sperdex-200 column, with PBS, Trisbuffer, or saline as the running solution. The peak fractions containinglarge molecular weight complex were collected and concentrated. Theprotein contents and the ratio of different antigens in MAPS complex wastested by SDS-PAGE with Coomassie blue staining, and theprotein/polysaccharide ratio of MAPS was determined by using BCA proteinassay kit and the anthrone assay.

Example 4 Immunization; Antibody and Cytokine Analysis; Challenge inMice

All immunogenic compositions and vaccines were prepared the day beforeimmunization. Pneumococcal whole cell vaccine, MAPS or an equimolarmixture containing all the specific antigens, rhizavidin, polysaccharideand biotin were diluted using saline to indicated concentration, andthen mixed with Al(OH)₃ in a 15 ml conical tube for adsorption overnightat 4° C.

C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me.) were used in allimmunization experiments. The age at time of first immunization wasbetween 4-6 weeks. Gently restrained, non-anesthetized mice received 3subcutaneous injections of 200 μl of adjuvant with or without indicatedamount of antigen in the lower part of the back at 2-week intervals.Blood was drawn 2 weeks after the second and/or the third immunization,and assayed for antibody and for cytokine production in vitro afterstimulation with pneumococcal whole cell antigen (WCA), TB extract, orparticular protein antigen.

Challenge was performed 2 weeks after the last immunization or bleeding.In NP colonization model, mice were intranasally challenged with 2×10⁷colony-forming units (CFU) of serotype 6B strain 0603 in 20 μl of PBS.To determine the presence and degree of NP colonization, an upperrespiratory culture was done 10 days later by instilling sterile salineretrograde through the transected trachea, collecting the first 6 drops(about 0.1 ml) from the nostrils, and plating neat or diluted samples onblood agar plates containing 2.5 μg gentamicin/ml.

In aspiration-sepsis challenge model, mice were gently anesthetized withisoflorane, held supine, and given a 100 μg intranasal inoculationcontaining 10⁶ CFU of pneumococci serotype 3 strain WU-2. Mice weremonitored twice daily and sacrificed by CO₂ inhalation and terminalexsanguination when demonstrating signs of illness, following which ablood culture was obtained.

Assays for murine antibodies to WCA or different protein antigens weredone in Immulon 2 HB 96-microwell plates (Thermo Scientific, Waltham,Mass.) coated with WCA (100 μg protein/ml PBS) or with protein antigens(1 μg of protein/ml PBS). Plates were blocked with 1% BSA in PBS.Antibody diluted in PBS-T was added and incubated at room temperaturefor 2 hr. Plates were washed with PBS-T, and secondary HRP-conjugatedantibody to mouse immunoglobulin G (from Sigma) was added and incubatedat room temperature for one hour. The plates were washed and developedwith SureBlue TMB Microwell Peroxidase Substrate (KPL, Gaithersburg,Md.).

For cytokine stimulation, the stimulants were diluted in stimulationmedium (DMEM (BioWhittaker, Walkersville, Md.) containing 10%low-endotoxin defined FBS (Hyclone, Logan, Utah), 50 μM2-mercaptoethanol (Sigma) and ciprofloxacin (10 μg/ml, Cellgro,Manassas, Va.)), at a concentration of 1 μg/ml-10 μg/ml for all proteinantigens, or for pneumococcal WCA. 25 μl of heparinized blood was addedto 225 μl DMEM medium with/without stimulants and cultured at 37° C. for6 days. Supernatants were collected following centrifugation and storedat −80° C. until analyzed by ELISA for IL-17A or IFN-γ concentration(R&D Systems, Minneapolis, Minn.).

For stimulation of splenocytes, mouse splenocytes were isolated,resuspended in stimulation medium, and then seeded in 48-well plate(3×10⁶ cells/well, in 300 μl of volume). After incubation at 37° C. for2 hr, stimuli were added at indicated concentration, for stimulation at37° C. for 3 days. Supernatants were collected following centrifugationand stored at −80° C. until analyzed by ELISA for IL-17A or IFN-γconcentration

Antibody and IL-17A concentrations and NP colonization densities werecompared by the Mann-Whitney U test using PRISM (version 4.0a forMacintosh, GraphPad Software, Inc). Differences in survival wereanalyzed with the Kaplan-Meier test, using PRISM as well.

We claim:
 1. An immunogenic composition comprising an immunologicallyeffective amount of at least one antigenic polysaccharide, animmunologically effective amount of one to ten different peptide orpolypeptide antigens, and one to ten different pairs of affinitymolecules, wherein each pair comprises a first affinity molecule and asecond affinity molecule complementary to the first affinity molecule,wherein in each pair: the first affinity molecule is associated with theat least one antigenic polysaccharide, and the complementary secondaffinity molecule is covalently attached to one of the peptide orpolypeptide antigens to form a fusion protein, and the first affinitymolecule non-covalently associates with the complementary secondaffinity molecule to link the respective peptide or polypeptide antigenand the at least one antigenic polysaccharide; and wherein theimmunogenic composition, upon administration to a subject, elicits (i)an immune response to the at least one antigenic polysaccharide, and(ii) an immune response to at least one of the one to ten differentpeptide or polypeptide antigens, in the subject.
 2. The immunogeniccomposition of claim 1, wherein the first affinity molecule iscross-linked or covalently bonded to the antigenic polysaccharide. 3.The immunogenic composition of claim 2, wherein the first affinitymolecule is cross-linked to the antigenic polysaccharide using across-linking reagent selected from any in the group consisting of: CDAP(1-cyano-4-dimethylaminopyridinium tetrafluoroborate); EDC(1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride); sodiumcyanoborohydride; cyanogen bromide; and ammonium bicarbonate/iodoaceticacid.
 4. The immunogenic composition of claim 1, wherein the pair(s) ofthe affinity molecules is/are each independently selected from the groupconsisting of: biotin/biotin-binding protein, antibody/antigen,enzyme/substrate, receptor/ligand, metal/metal-binding protein,carbohydrate/carbohydrate binding protein, lipid/lipid-binding protein,and His tag/His tag-binding substance.
 5. The immunogenic composition ofclaim 1, wherein the first affinity molecule is biotin or a derivativeor mimic molecule thereof.
 6. The immunogenic composition of claim 1,wherein the complementary second affinity molecule is a biotin-bindingprotein, or an avidin-like protein or a derivative or functional portionthereof.
 7. The immunogenic composition of claim 6, wherein theavidin-like protein is selected from the group consisting of:rhizavidin, avidin, streptavidin, and a homologue or derivative thereof.8. The immunogenic composition of claim 1, wherein the antigenicpolysaccharide is a branched chain polysaccharide or a straight chainpolysaccharide.
 9. The immunogenic composition of claim 1, comprisingtwo to ten different peptide or polypeptide antigens, wherein thepeptide or polypeptide antigens are selected from the group consistingof: different peptides or polypeptides; different variants of the samepeptide or polypeptide; and different domains or portions of the samepeptide or polypeptide.
 10. The immunogenic composition of claim 1,further comprising a flexible linker peptide attached to the peptide orpolypeptide antigens, wherein the flexible linker peptide attaches thepeptide or polypeptide antigens to the complementary second affinitymolecule.
 11. The immunogenic composition of claim 1, wherein at leastone of the peptide or polypeptide antigen is from a pathogenic organism,or a cancer or tumor.
 12. The immunogenic composition of claim 1,wherein at least one of the peptide or polypeptide antigens is selectedfrom the group consisting of: tuberculosis antigens; Staphylococcusaureus antigens; Acinetobacter antigens; enteric Gram-negative bacterialantigens; nonenteric Gram-negative bacterial antigens; Gram-positivebacterial antigens; toxoids, toxins or toxin portions; fungal antigens;viral antigens; cancer or tumor antigens; and combinations thereof. 13.The immunogenic composition of claim 1, wherein the antigenicpolysaccharide is selected from the group consisting of:polysaccharides, oligosaccharides, or lipopolysaccharides fromGram-positive bacteria; polysaccharides, oligosaccharides, orlipopolysaccharides from Gram-negative bacteria; other bacterialcapsular or cell wall polysaccharides; fungal polysaccharides; viralpolysaccharides; and polysaccharides derived from cancer or tumor cells.14. The immunogenic composition of claim 1, further comprising at leastone co-stimulation factor associated with the antigenic polysaccharideor at least one of the peptide or polypeptide antigens.
 15. Theimmunogenic composition of claim 14, wherein the co-stimulation factoris selected from the group consisting of: a Toll-like receptorligand/agonist, a NOD ligand/agonist, and an activator/agonist of aninflammasome.
 16. The immunogenic composition of claim 1, furthercomprising at least one adjuvant.
 17. A method for inducing an immuneresponse in a subject to at least one antigen, comprising administeringto the subject a composition of claim
 1. 18. The method of claim 17,wherein the immune response is an antibody or B cell response.
 19. Themethod of claim 17, wherein the immune response is a CD4+ T cellresponse, including Th1, Th2, or Th17 response, or a CD8+ T cellresponse, or CD4+/CD8+ T cell response.
 20. The method of claim 17,wherein the immune response is: an antibody or B cell response; and a Tcell response.
 21. The method of claim 17, wherein the immune responseis to at least one antigenic polysaccharide or at least one peptide orpolypeptide antigen.
 22. The method of claim 17, wherein the immuneresponse is an antibody or B cell response to at least one antigenicpolysaccharide and a CD4+ T cell response, including Th 1, Th2, or Th 17response, or a CD8+ T cell response, or CD4+/CD8+ T cell response to atleast one peptide or polypeptide antigen.
 23. The method of claim 17,wherein the immune response is an antibody or B cell response to atleast one antigenic polysaccharide, and an antibody or B cell responseand a CD4+ T cell response, including Th1, Th2, or Th 17 response, or aCD8+ T cell response, or CD4+/CD8+ T cell response to at least onepeptide or polypeptide antigen.
 24. The immunogenic composition of claim12, wherein the enteric Gram-negative bacterial antigens are selectedfrom the group of: E. coli antigens, Salmonella antigens, Enterobacterantigens, Klebsiella antigens, Citrobacter antigens, Serratia antigens,Clostridia antigens, Shigella antigens, Campylobacter antigens, Vibriocholera antigens, and combinations thereof.
 25. The immunogeniccomposition of claim 12, wherein the nonenteric Gram-negative bacterialantigens are selected from the group of: Pertussis antigens,Meningococcal antigens, Haemophilus antigens, Pseudomonas antigens, andcombinations thereof.
 26. The immunogenic composition of claim 12,wherein the Gram-positive bacterial antigens are pneumococcal antigensor anthrax antigens.
 27. The immunogenic composition of claim 12,wherein the viral antigens are HIV antigens, or seasonal or epidemicinfluenza antigens.
 28. The immunogenic composition of claim 13, whereinthe antigenic polysaccharide is selected from the group consisting of:Salmonella typhi Vi capsular polysaccharides; Salmonellapolysaccharides; pneumococcal polysaccharides; Haemophilipolysaccharides; Meningococcal polysaccharides; Staphylococcus aureuspolysaccharides; Bacillus anthracis polysaccharides; Streptococcuspolysaccharides; Pseudomonas polysaccharides; Cryptococcuspolysaccharides; and viral glycoproteins.
 29. The immunogeniccomposition of claim 28, wherein the antigenic polysaccharide isselected from the group consisting of: Salmonella typhi Vi capsularpolysaccharides; pneumococcal capsular polysaccharides; pneumococcalcell wall polysaccharides; Staphylococcus aureus capsularpolysaccharides; Haemophilus influenzae Type b (Hib) polysaccharides; GpA Streptococcus polysaccharides; and Gp B Streptococcus polysaccharides.30. The immunogenic composition of claim 1, comprising five to tendifferent peptide or polypeptide antigens, wherein the peptide orpolypeptide antigens are selected from the group consisting of:different peptides or polypeptides; different variants of the samepeptide or polypeptide; and different domains or portions of the samepeptide or polypeptide.
 31. The immunogenic composition of claim 1,comprising two to ten different peptide or polypeptide antigens, whereinat least two different peptide or polypeptide antigens are linked to thesame antigenic polysaccharide.
 32. The immunogenic composition of claim1, comprising at least two different antigenic polysaccharides and twoto ten different peptide or polypeptide antigens, wherein at least twodifferent peptide or polypeptide antigens are each linked to a differentantigenic polysaccharide.
 33. The immunogenic composition of claim 1,wherein the complementary second affinity molecule is covalentlyattached to a fusion protein comprising two or more peptide orpolypeptide antigens.
 34. An immunogenic composition comprising: animmunologically effective amount of at least one antigenicpolysaccharide, and an immunologically effective amount of one to tendifferent peptide or polypeptide antigens, wherein the at least oneantigenic polysaccharide is cross-linked or covalently bonded to biotin,and the one to ten peptide or polypeptide antigens are associated withat least one rhizavidin peptide, derivative, or functional portionthereof, and wherein the one to ten peptide or polypeptide antigens andthe at least one antigenic polysaccharide are linked by a non-covalent,affinity interaction between the biotin and the rhizavidin peptide,derivative, or functional portion thereof; and wherein the immunogeniccomposition, upon administration to a subject, elicits (i) an immuneresponse to the at least one antigenic polysaccharide, and (ii) animmune response to at least one of the one to ten different peptide orpolypeptide antigens, in the subject.
 35. The immunogenic composition ofclaim 1, wherein the immune response to the at least one antigenicpolysaccharide and/or to at least one of the one to ten differentpeptide or polypeptide antigens comprises an antibody or B cellresponse.
 36. The immunogenic composition of claim 1, wherein the immuneresponse to the at least one antigenic polysaccharide and/or to at leastone of the one to ten different peptide or polypeptide antigenscomprises a T cell response.
 37. The immunogenic composition of claim 1,wherein the immune response to the at least one antigenic polysaccharideand/or to at least one of the one to ten different peptide orpolypeptide antigens comprises a CD4+ T cell response, including Th1,Th2, or Th17 response, or a CD8+ T cell response, or a CD4+/CD8+ T cellresponse.
 38. The immunogenic composition of claim 1, wherein the immuneresponse to the at least one antigenic polysaccharide and/or to at leastone of the one to ten different peptide or polypeptide antigenscomprises: an antibody or B cell response; and a T cell response. 39.The immunogenic composition of claim 1, wherein the immunogeniccomposition, upon administration to a subject, elicits at least (i) anantibody or B cell response to the at least one antigenicpolysaccharide, and (ii) an antibody or B cell response to at least oneof the one to ten different peptide or polypeptide antigens, in thesubject.
 40. The immunogenic composition of claim 1, wherein theimmunogenic composition, upon administration to a subject, elicits atleast (i) an antibody or B cell response to the at least one antigenicpolysaccharide, and (ii) an antibody or B cell response and a CD4+ Tcell response, including Th1, Th2, or Th17 response, or a CD8+ T cellresponse, or a CD4+/CD8+ T cell response to at least one of the one toten different peptide or polypeptide antigen, in the subject.
 41. Theimmunogenic composition of claim 1, wherein the immunogenic composition,upon administration to a subject, elicits at least (i) an antibody or Bcell response to the at least one antigenic polysaccharide, and (ii) aCD4+ T cell response, including Th1, Th2, or Th17 response, or a CD8+ Tcell response, or a CD4+/CD8+ T cell response to at least one of the oneto ten different peptide or polypeptide antigens, in the subject. 42.The method of claim 17, further comprising administering to the subjecta therapeutic agent.